High EGFR mrna expression is a prognostic factor for reduced survival in pancreatic cancer after gemcitabine-based adjuvant chemotherapy

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1 INTERNATIONAL JOURNAL OF ONCOLOGY 38: , 2011 High EGFR mrna expression is prognostic fctor for reduced survivl in pncretic cncer fter gemcitbine-bsed djuvnt chemotherpy Hyto Fujit 1, Kenoki Ohuchid 1,2, Kzuhiro Mizumoto 1,3, Soichi Itb 4, Tetsuhide Ito 4, Kohei Nkt 1,5, Jun Yu 1, Tdshi Kyshim 1, Akifumi Hyshi 1,5, Ryot Souzki 6, Ttsuro Tjiri 6, Mnbu Onimru 1, Ttsuy Mnbe 1, Tko Ohtsuk 1 nd Mso Tnk 1 Deprtments of 1 Surgery nd Oncology, nd 2 Advnced Medicl Inititives, Grdute School of Medicl Sciences, Kyushu University; 3 Kyushu University Hospitl Cncer Center, Midshi; Deprtments of 4 Medicine nd Bioregultory Science, 5 Antomic Pthology, nd 6 Peditric Surgery, Grdute School of Medicl Sciences, Kyushu University, Midshi, Fukuok , Jpn Received October 26, 2010; Accepted December 20, 2010 DOI: /ijo Abstrct. Pncretic ductl denocrcinom (PDAC) still presents mjor therpeutic chllenge nd phse III clinicl tril hs reveled tht the combintion of gemcitbine nd humn epiderml growth fctor receptor type I (HER1/ EGFR) trgeting gent presented significnt benefit compred to tretment with gemcitbine lone. The im of this study ws to investigte EGFR mrna expression in resected PDAC tissues nd its correltion with ptient prognosis. We obtined formlin-fixed prffin-embedded (FFPE) tissue smples from 88 ptients with PDAC who underwent pncretectomy, nd mesured EGFR mrna levels by quntittive rel-time reverse trnscription-polymerse chin rection. The highlevel EGFR group hd significntly shorter disese-free-survivl (p=0.029) nd overll-survivl (p=0.014) s shown by univrite nlyses, lthough these did not rech sttisticl significnce, s shown by multivrite nlyses. However, we found tht high EGFR expression ws n independent prognostic fctor in ptients receiving gemcitbine-bsed djuvnt chemotherpy (p=0.023). Furthermore, we mesured Correspondence to: Dr Kenoki Ohuchid or Dr Kzuhiro Mizumoto, Deprtment of Surgery nd Oncology, Grdute School of Medicl Sciences, Kyushu University, Midshi, Fukuok , Jpn E-mil: kenoki@med.kyushu-u.c.jp E-mil: mizumoto@med.kyushu-u.c.jp Key words: epiderml growth fctor receptor, pncretic cncer, reverse trnscription-polymerse chin rection, formlin-fixed prffin-embedded smples, endoscopic ultrsound-guided fine needle spirtion EGFR mrna levels in 20 endoscopic ultrsound-guided fine needle spirtion (EUS-FNA) cytologicl specimens. Altered EGFR levels were distinguishble in microdissected neoplstic cells from EUS-FNA cytologicl specimens compred to those in whole cell pellets. In conclusion, quntittive nlysis of EGFR mrna expression using FFPE tissue smples nd microdissected neoplstic cells from EUS-FNA cytologicl specimens could be useful in predicting prognosis nd sensitivity to gemcitbine in PDAC ptients. Introduction Pncretic ductl denocrcinom (PDAC) is one of the most lethl nd ggressive humn mlignncies, nd it is the fourth leding cuse of tumor-relted deths in the industrilized world (1,2). The vst mjority of ptients with PDAC hve poor outcomes due to the ggressive nture of the tumor nd difficulties in erly dignosis due to the lck of erly disesespecific signs nd symptoms. Only 10-20% of ptients with PDAC hve chnce of curtive resection (3) nd, even if the curtive resection is performed, the post-opertive 5-yer survivl rte is only 15-25% due to high recurrence rte (4,5). Two rndomized clinicl phse III trils of djuvnt chemotherpy (AC) for PDAC hve shown significnt increses in overll survivl (OS) nd disese-free survivl (DFS). However, their efficcy ws limited nd insufficient (6,7). To improve the prognosis of ptients with PDAC, individulized chemotherpy bsed on the gene expression profiles of the individul's own cncer tissues, could be potent strtegy. Humn epiderml growth fctor receptor type 1 (HER1/ EGFR) is receptor tyrosine kinse. Binding of lignd growth fctors, such s epiderml growth fctor (EGF) nd trnsforming growth fctor (TGF)-α to EGFR leds to receptor phosphoryltion nd ctivtion of downstrem Rs/mitogenctivted protein kinse (MAPK) signling, thereby enhncing the mlignnt behvior of cncer cells (8,9). There is incresing

2 630 fujit et l: EGFR mrna expression in PDAC evidence showing tht the dysregultion of EGFR pthwys by overexpression or constitutive ctivtion cn promote tumor growth nd metstsis, nd tht this is ssocited with poor prognosis nd tumor ggressiveness in mny humn mlignncies, including pncretic cncer (10-13). To improve the prognosis of PDAC ptients, the blockde of the EGFR signling pthwy could be potent strtegy (9,14). The EGFR signling blockde hs been reported to decrese growth nd metstsis in n orthotopic implnttion murine model of pncretic cncer cells (15) nd to improve the efficcy of gemcitbine in humn pncretic tumor xenogrft models (16). At the time of dignosis, >80% of PDAC ptients present with either loclly dvnced or metsttic disese (3). Therefore, ptients with unresectble dvnced PDAC require cytopthologicl ssessment using endoscopic ultrsoundguided fine needle spirtion (EUS-FNA) or pncretic juice specimens to predict their sensitivity to chemotherpeutic gents nd prognosis. Quntittive mrna nlysis of genes ssocited with sensitivity to chemotherpeutic gents, or with ptient prognosis could be suitble for clinicl use s this method enbles us to reproducibly detect gene expression, even with smll smples (17). In the current study, we investigted the correltion between EGFR expression nd the prognosis of ptients with PDAC. To elucidte the role of EGFR expression in gemcitbine sensitivity, we lso investigted the ssocition between receptor expression levels nd tretment outcomes in PDAC ptients receiving gemcitbinebsed AC. Furthermore, we quntified EGFR expression in cytologicl specimens obtined by EUS-FNA to exmine the possible utility of such smples for quntifying the mrna levels of these predictive fctors. Mterils nd methods Ptients nd pncretic tissues. Our study subjects comprised of 88 ptients who underwent pncretectomy for PDAC t the Deprtment of Surgery nd Oncology, Kyushu University Hospitl (Fukuok, Jpn) from 1992 to The ptients (54 mle nd 34 femle) hd medin ge of 65 yers (rnge, yers). Eighteen of the 88 ptients received no AC (non-ac group). Thirty-six of the 88 ptients received gemcitbine-bsed AC (GEM group), consisting of two or more cycles of 1,000 mg/m 2 /d gemcitbine on dys 1, 8 nd 15 every 28 dys, nd three or more cycles of 1,000 mg/m 2 /d gemcitbine on dys 1 nd 8 every 21 dys. Nineteen of the 88 ptients received other forms of AC (other AC group), including 5 ptients orlly dministered S-1 ( mg/ body), 7 ptients orlly dministered tegfur ( mg/ body), nd 7 ptients treted with bolus of 5-fluorourcil ( mg/body). The remining 15 ptients did not receive dequte AC due to their poor performnce sttus. We recommended tht ptients hd follow-up visits every 3 months for 2 yers, then visits every 6 months for 3 yers, nd then nnul visits. DFS ws defined s the time from the dte of pncretic resection to the dte of locl or distnt recurrence. The dte of recurrence ws defined s the dte of the first subjective symptom herlding relpse, or the dte of documenttion of recurrent disese, independent of site, s ssessed by dignostic imging techniques (whichever occurred first). Dt for Tble I. Clinicopthologicl chrcteristics of the ptients (n=88). Medin ge 65 yers (rnge, yers) Gender (mle/femle) 54 (61.4%)/34 (38.6%) Histologicl dignosis Adenocrcinom 86 (97.7%) Adenosqumous crcinom 2 (2.3%) Adjuvnt chemotherpy (AC) No 18 (20.5%) Yes 55 (62.5%) Gemcitbine-bsed AC 36 (40.9%) Other AC 19 (21.6%) Rdiotherpy including IOR Yes 23 (26.1%) No 53 (60.2%) pt ctegory pt1 5 (5.8%) pt2 3 (3.4%) pt3 78 (89.7%) pt4 1 (1.1%) pn ctegory pn0 27 (31.0%) pn1 60 (69.0%) UICC stge I 6 (6.9%) II 78 (89.7%) III 1 (1.1%) IV 2 (2.3%) Histologicl grde G1 20 (23.3%) G2 33 (38.4%) G3 33 (38.4%) Residul tumor ctegory R0 55 (63.9%) R1 31 (36.1%) Vessel invsion Positive 55 (63.2%) Negtive 32 (36.8%) Neurl invsion Positive 72 (82.8%) Negtive 15 (17.2%) ptients without recurrence were censored t the time of the lst follow-up visit. OS ws mesured from the dte of pncretic resection to the dte of deth. Fifty-eight ptients died during follow-up nd the other ptients were censored t the time of the lst follow-up visit. Dt were nlyzed in December 2009 nd follow-up dt from ll cses were vilble. The medin observtion time for DFS ws 9 months (rnge, months) nd OS ws 18 months (rnge, months). The clinicopthologicl chrcteristics of the tumors collected from 88 ptients re provided in Tble I. Additionlly,

3 INTERNATIONAL JOURNAL OF ONCOLOGY 38: , in order to compre the EGFR expression levels in PDAC tissues to those in non-mlignnt pncretic specimens, totl of 40 non-mlignnt pncretic tissues, including 10 norml pncretic tissues resected with bile duct crcinom nd 30 chronic pncretitis tissues, were lso obtined. All resected specimens were fixed in formlin nd embedded in prffin, nd ll tissues djcent to the specimens were evluted histologiclly ccording to the criteri of the World Helth Orgniztion. Two pthologists were in greement s regrds the pthologicl fetures of ll cses nd the dignoses were confirmed. The tumor stge ws ssessed ccording to the Union Interntionle Contre le Cncer (UICC) nd the Americn Joint Committee on Cncer guidelines (18). The study ws pproved by the Ethics Committee of Kyushu University nd conducted ccording to the Ethicl Guidelines for Humn Genome/Gene Reserch encted by the Jpnese Government nd the Helsinki Declrtion. Immunohistochemistry. A totl of 25 sections (4-µm thick) from formlin-fixed prffin-embedded (FFPE) specimens from 88 ptients with PDAC nd 15 sections from 40 nonmlignnt cses, including seven sections from norml pncres resected with bile duct crcinom, nd 8 sections from chronic pncretitis ptients, were deprffinized in xylene nd hydrted in grded ethnol. Endogenous peroxidse ctivity ws blocked by incubtion with 3% hydrogen peroxide in methnol for 30 min. Antigen retrievl ws chieved by utoclving the sections in citrte buffer t ph 6.0. The Histofine SAB-PO(R) kit (Nichirei, Tokyo, Jpn) ws used for immunohistochemicl lbeling. The sections were incubted with 1.5% norml got serum/phosphte-buffered sline, followed by incubtion with rbbit polyclonl nti- EGFR ntibody (Snt Cruz Biotechnology, Snt Cruz, CA) t 1:50 dilution overnight t 4 C. The sections were incubted with biotinylted nti-rbbit immunoglobulin solution for 20 min followed by peroxidse-lbeled streptvidin for 20 min. Immunocomplexes were visulized using stble 3,3'- diminobenzidine tetrhydrochloride (Dojin, Kummoto, Jpn). The sections were rinsed with distilled wter nd counterstined with hemtoxylin for 10 sec. The mount of EGFR immunorectivity ws evluted using the following scle ccording to the percentge of EGFR-positive cncer cells: 0, <5%; 1, 5 25%; 2, 26 50%; nd 3, >51%. Stining intensity ws scored semi-quntittively s follows: 0, bsent; 1, wek; 2, moderte; 3, strong. To perform the quntittive nlysis of EGFR immunorectivity, the following combined score ws determined: Degree of stining = quntity x intensity. We lso performed dditionl stining without primry ntibodies s the negtive control. All slides were evluted independently by three investigtors (H.F., A.H. nd K.N.) without ny knowledge of the bckground of ech cse. Cytologicl specimens. Cytologicl specimens were obtined t the time of cytologicl exmintion nd dignosis from the pthologicl lbortory of Kyushu University Hospitl. In brief, cytologicl specimens were divided into whole cell pellets (WCP) nd into three or more smers s soon s possible fter retrievl. Smers were processed in three different wys s described previously (17). Two smers were mounted on stndrd glss slides for Hemcolor stining (Merck KGA, Drmstdt, Germny) nd Ppnicolou stining, then used for rpid cytologicl dignosis nd strict cytologicl dignosis, respectively. These two smers were exmined histologiclly by cytopthologists nd dignosis ws confirmed ccording to the Ppnicolou Clssifiction. The third smer of ech specimen ws mounted on membrne slides (Leic Microsystems, Wetzlr, Germny) for lser cpture microdissection (LCM). These smers were stined in 1% toluidine blue stining solution or by Hemcolor stining. Twenty cytologicl specimens were obtined from ptients t the Kyushu University Hospitl who underwent EUS-FNA cytology nd who were cytopthologiclly dignosed with PDAC. Isoltion of RNA. Totl RNA ws isolted from FFPE tissue smples using the RNesy FFPE kit (Qigen, Tokyo, Jpn) with some modifiction to the mnufcturer's instructions fter mcrodissection bsed on review of representtive hemtoxylin nd eosin-stined slides s described previously (19). Totl RNA ws extrcted from cells isolted by microdissection ccording to the stndrd cid gunidinium thiocynte-phenol-chloroform protocol (20), with or without glycogen (Funkoshi, Tokyo, Jpn). Quntittive rel-time reverse trnscription-polymerse chin rection (qrt-pcr). qrt-pcr ws performed using the Chromo4 Rel-Time PCR Detection System (Bio-Rd Lbortories, Hercules, CA, USA) nd the LightCycler 480 II Rel-Time PCR System (Roche Dignostics) for 40 cycles of 15 sec t 95 C nd 1 min t 55 C with the QuntiTect SYBR- Green Reverse Trnscription-PCR kit (Qigen) in ccordnce with the mnufcturer's instructions (21). We designed specific primers for EGFR (forwrd primer, 5'-ccttgtgcggg tttgtcttt-3'; nd reverse primer, 5'-ccctgtgttggggctg-3') nd β-ctin (forwrd primer, 5'-tggcgcggctcgctt-3'; nd reverse primer, 5'-tcctttgtccgccgttt-3'), nd screened dtbse using BLASTN to confirm the primer specificities. The level of ech mrna ws clculted from stndrd curve constructed using totl RNA from Cpn-1, humn pncretic cncer cell line. The level of EGFR mrna ws normlized to tht of β-ctin. The PCR product sizes of EGFR nd β-ctin primers were smll [88 bse pirs (bp) nd 59 bp, respectively], which llowed for ccurte nd sensitive qrt-pcr nlysis despite the frgmented RNA extrcted from the FFPE tissue specimens (22,23). Sttisticl nlyses. Sttisticl nlyses nd grphicl presenttions were performed using JMP 7.01 softwre (SAS Institute, Cry, NC, USA). Vlues were expressed s the mens ± SD. Dt were nlyzed using the Kruskl-Wllis test if comprisons involved three groups, nd the Mnn-Whitney U-test nd Spermn's rnk-correltion test if comprisons involved two groups s norml distributions were not obtined. EGFR expression ws split into high- nd low-level groups using recursive descent prtition nlysis, s described by Hoffmnn et l (24). Ctegoricl vribles were compred using the χ 2 test (Fisher's exct probbility test). Survivl curves were constructed using the Kpln-Meier product-limit method nd were compred using the log-rnk test. To evlute independent prognostic fctors ssocited with survivl, multivrite Cox

4 632 fujit et l: EGFR mrna expression in PDAC Figure 1. Immunohistochemicl nlysis of EGFR in norml pncres, chronic pncretitis nd PDAC tissues. Wek to moderte immunorectivity for EGFR ws detected in some cinr cells nd pncretic ductl cells (A-D). In PDAC tissues, immunorectivity for EGFR ws observed on the surfce nd in the cytoplsm of cncer cells (E-G), with no immunorectivity in the surrounding strom (E-H). The immunorectivity ws different in respective cses (E, strong; F, moderte; G, wek expression; H, bsent). Scle brs represent 200 µm (A, C, E-H) nd 100 µm (B nd D). proportionl hzrds regression nlysis ws used. Sttisticl significnce ws defined s p-vlue of <0.05. Results EGFR protein expression ws correlted with EGFR mrna expression. We performed immunohistochemicl nlyses on 15 sections of non-mlignnt pncretic tissues, including 7 norml, 8 chronic pncretitis tissues nd 25 PDAC tissues. In greement with the findings of previous studies (10,13), wek to moderte immunorectivity for EGFR ws detected in some cinr cells nd pncretic ductl cells (Fig. 1A-D). EGFR immunorectivity ws observed on the surfce nd in the cytoplsm of cncer cells within PDAC tissues, but none ws observed in the surrounding strom (Fig. 1E-G) (10,13). To investigte the correltion between EGFR immunorectivity nd EGFR mrna expression levels within ech FFPE tissue smple from resected PDAC tissue, we evluted the degree of stining (quntity x intensity) for n nti-egfr ntibody, s the immunorectivity ws different in respective cses (Fig. 1E, strong; F, moderte; G, wek expression; nd H, bsent). We found significnt correltion between the degree of stining nd EGFR mrna expression levels [Fig. 2A; Spermn's rnk-correltion coefficient (ρ): 0.729, p<0.0001], nd cses with higher degree of immunorectivity expressed significntly higher levels of EGFR mrna compred with those with lower degree of immunorectivity (Fig. 2B; p=0.0005). These observtions suggest tht quntittive mrna nlysis of EGFR in mcrodissected PDAC tissues my reflect EGFR protein expression levels in EGFR-

5 INTERNATIONAL JOURNAL OF ONCOLOGY 38: , Figure 3. Quntittive nlysis of EGFR mrna expression levels in FFPE tissue smples from resected PDAC tissues (n=88) using qrt-pcr. After normliztion to β-ctin expression, we obtined 2 groups with high EGFR expression nd low EGFR expression using cut-off vlue (0.058) determined with recursive descent prtition nlysis, respectively (A). There ws no significnt difference in EGFR mrna levels between the respective groups (non-ac group, n=18; GEM group, n=36; other AC group, n=19; p=0.26) (B). Figure 2. Correltion between EGFR immunorectivity nd EGFR mrna expression levels in ech FFPE tissue smple from resected PDAC tissues (A; n=25). We observed significnt correltion between the degree of stining (quntity x intensity) nd EGFR mrna expression levels [A; Spermn's rnk-correltion coefficient (ρ): 0.729, p<0.0001], nd cses with higher degree of stining (6-9; n=15) expressed significntly higher levels of EGFR mrna compred to cses with lower degree of stining (0-4; n=10) (B; p=0.0005). We found tht EGFR expression levels in the PDAC smples (n=88) were significntly higher thn those in the non-mlignnt smples (n=40) (C; p=0.0004). expressing cncer cells. Additionlly, lthough there ws immunorectivity for EGFR in some cinr nd ductl cells in non-mlignnt cses, we found tht EGFR expression levels in PDAC smples (n=88) were significntly higher thn those in non-mlignnt smples (n=40) (Fig. 2C; p=0.0004). Univrite nd multivrite nlyses of EGFR mrna expression nd survivl time. We quntified EGFR mrna expression levels in FFPE tissue smples from resected PDAC tissues using qrt-pcr. After normliztion to β-ctin, we obtined two groups (high EGFR expression nd low EGFR expression) using cut-off vlue (0.058) determined by recursive descent prtition nlysis of ll ptients (n=88) (Fig. 3A) (24). The high nd low EGFR expression groups comprised of 46 nd 42 cses, respectively. The reltionship between EGFR mrna expression nd the clinicopthologicl fctors seen in PDAC ptients is shown in Tble II. We found no significnt correltion between EGFR mrna expression nd clinicopthologicl fctors. In ddition, there ws no significnt difference in EGFR mrna levels between the non-ac group, the GEM group, nd the other AC group (p=0.26; Fig. 3B). Initilly, we exmined the independent mrkers tht indicted poor prognosis in the 88 PDAC ptients. Univrite nlyses for DFS nd OS (Tble III) showed tht conventionl prognostic mrkers, such s pn sttus (p= nd p=0.0026, respectively), residul tumor ctegory (R fctor) (p< nd p<0.0001, respectively), nd positive vessel invsion (p= nd p=0.0035, respectively) reched sttisticl significnce, wheres the effect of AC did not (p=0.23 nd p=0.066, respectively). High EGFR levels fter normliztion to β-ctin were ssocited with shorter DFS nd OS (Tble III nd Fig. 4A-B; p=0.029 nd p=0.014, respectively). Multivrite nlysis bsed on the Cox proportionl hzrds model ws performed on ll prmeters tht were found to be significnt by univrite nlyses for DFS (Tble IV) nd OS

6 634 fujit et l: EGFR mrna expression in PDAC Tble II. Reltionship between EGFR mrna expression nd vrious clinicopthologicl fctors. EGFR mrna expression Chrcteristics High-level group (n=46) Low-level group (n=42) P-vlue Age yers 24 (52.2%) 23 (54.8%) <65 yers 22 (47.8%) 19 (45.2%) Adjuvnt chemotherpy (AC) No 8 (17.4%) 10 (23.8%) Yes 33 (71.7%) 22 (52.4%) Gemcitbine-bsed AC 21 (45.7%) 15 (35.7%) Other AC 12 (26.1%) 7 (16.7%) Rdiotherpy Yes 10 (21.7%) 13 (30.9%) No 29 (63.0%) 24 (57.1%) pt ctegory pt1/pt2 5 (10.9%) 3 (7.1%) pt3/pt4 41 (89.1%) 39 (92.9%) pn ctegory pn0 11 (23.9%) 16 (38.1%) pn1 34 (73.9%) 26 (61.9%) UICC stge I 3 (6.5%) 3 (7.1%) II 41 (89.1%) 37 (88.1%) III/IV 1 (2.2%) 2 (4.8%) Histologicl grde G1 10 (21.7%) 10 (23.8%) G2 14 (30.4%) 19 (45.2%) G3 21 (45.7%) 12 (28.6%) Residul tumor ctegory R0 26 (56.5%) 29 (69.0%) R1 18 (39.1%) 13 (30.9%) Vessel invsion Positive 31 (67.4%) 24 (57.1%) Negtive 14 (30.4%) 18 (42.9%) Neurl invsion Positive 37 (80.4%) 35 (83.3%) Negtive 8 (17.4%) 7 (16.7%) Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88). (Tble V). DFS ws significntly dependent on the R fctor (p<0.0001) nd vessel invsion (p=0.038), wheres OS ws significntly dependent on the R fctor lone (p<0.0001). The effect of high EGFR levels did not rech sttisticl significnce for either DFS or OS. In order to determine which prmeters were predictive for gemcitbine sensitivity, we then evluted the correltion between ech prmeter nd DFS in the GEM nd non-ac groups. We found no significnt correltion between the level of EGFR mrna expression nd clinicopthologicl fctors in the GEM group (Tble VI). Univrite survivl nlyses of the GEM group showed tht pn sttus (p=0.0094), residul tumor (p=0.0004), nd high EGFR level normlized to β-ctin (Fig. 5A; p=0.068) reched sttisticl significnce for DFS (Tble VII). However, there ws no significnt correltion between the EGFR expression level nd DFS in the non-ac group (p=0.30, Fig. 5C), lthough the number of ptients who did not receive AC ws limited. Multivrite nlysis of the GEM group (Tble VIII) showed tht DFS ws significntly dependent on both the R fctor (p=0.0071) nd high EGFR levels (p=0.010). Similrly, we evluted the correltion between ech prmeter nd OS in the GEM nd non-ac groups. Univrite survivl nlyses of the GEM group showed tht the conventionl prognostic mrkers, pn sttus (p=0.020), R fctor (p=0.013), nd high EGFR levels normlized to β-ctin (Fig. 5B; p=0.054) reched sttisticl significnce for OS (Tble VII). However, the effect of EGFR expression levels

7 INTERNATIONAL JOURNAL OF ONCOLOGY 38: , Tble III. Univrite survivl nlysis of conventionl prognostic fctors nd EGFR mrna expression (n=88). Chrcteristics Number of Medin DFS P-vlue Medin OS P-vlue 5-yer survivl cses (months) (months) rte EGFR mrna expression b b High % Low % Age yers % <65 yers % Adjuvnt chemotherpy (AC) Yes % No % Rdiotherpy Yes % No % pt ctegory pt1/pt % pt3/pt % pn ctegory b b pn % pn % UICC stge I % II % III/IV % Histologicl grde G1/G % G % Residul tumor ctegory <0.001 b <0.001 b R % R % Vessel invsion b b Positive % Negtive % Neurl invsion Positive % Negtive % Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88); b p<0.05. Tble IV. Multivrite DFS nlysis (Cox regression model) of conventionl prognostic fctors nd EGFR mrna. Chrcteristics Reltive risk 95% Confidence intervl P-vlue High EGFR levels pn ctegory (pn1) Residul tumor ctegory (pr1) < b Positive vessel invsion b Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88); b p<0.05.

8 636 fujit et l: EGFR mrna expression in PDAC Figure 4. DFS nd OS fter resection of PDAC with high versus low EGFR expression. High EGFR mrna levels were ssocited with shorter DFS (A, p=0.029) nd shorter OS (B, p=0.014). * p<0.05. did not rech significnce in the non-ac group (p=0.07, Fig. 5D). Multivrite nlysis of the GEM group (Tble IX) showed tht OS ws significntly dependent on pn sttus (p=0.024), R fctor (p=0.045), nd high EGFR levels (p=0.023). These dt suggest tht high EGFR expression is significnt predictor for reduced DFS nd significnt prognostic indictor for reduced OS, especilly in those ptients receiving gemcitbine-bsed AC. Quntittive nlysis of EGFR expression in cells microdissected from cytologicl specimens. In order to pply this prediction of outcome for PDAC ptients receiving gemcitbinebsed chemotherpy bsed on EGFR expression levels to clinicl setting, we quntified the EGFR mrna levels in cytologicl specimens obtined from 20 ptients with PDAC who underwent EUS-FNA cytologicl exmintion t our institute. Although some smples contined bundnt Figure 5. DFS nd OS fter resection of PDAC with high versus low EGFR expression in the GEM (A nd B; n=36) nd non-ac (C nd D; n=18) groups. High EGFR mrna levels were ssocited with shorter DFS (A, p=0.068) nd shorter OS (B, p=0.054) in the GEM group. In contrst, there ws no significnt correltion between EGFR expression levels nd DFS (C, p=0.30) or OS (D, p=0.07) in the non-ac group. * p<0.05. neoplstic cells, most smples contined lrge mount of blood nd inflmmtory cells nd scrce clusters of neoplstic cells (Fig. 6A-B). Therefore, we quntified the EGFR mrna levels in the WCP nd LCM smples, nd then compred the expression levels between the two. We were unble to detect cler differences in EGFR mrna levels in the WCP smples. However, we distinguished higher nd lower expression levels of the mrna in the LCM smples (Fig. 6C). These dt suggest tht the quntifiction of EGFR expression levels in microdissected neoplstic cells could be potent tool for Tble V. Multivrite OS nlysis (Cox regression model) of conventionl prognostic fctors nd EGFR mrna. Chrcteristics Reltive risk 95% Confidence intervl P-vlue High EGFR levels (>0.058) pn ctegory (pn1) Residul tumor ctegory (pr1) < b Positive vessel invsion Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88); b p<0.05.

9 INTERNATIONAL JOURNAL OF ONCOLOGY 38: , Tble VI. Reltionship between EGFR mrna expression nd vrious clinicopthologicl fctors in the GEM group (n=36). EGFR mrna expression Chrcteristics High-level group (n=46) Low-level group (n=42) P-vlue Age yers 11 (52.4%) 4 (26.7%) <65 yers 10 (47.6%) 11 (73.3%) Gender Mle 14 (66.7%) 9 (60.0%) Femle 7 (33.3%) 6 (40.0%) Rdiotherpy Yes 2 (9.5%) 5 (33.3%) No 18 (90.5%) 8 (66.7%) pt ctegory pt1/pt2 1 (4.8%) 0 (0%) pt3/pt4 20 (95.2%) 15 (100%) pn ctegory pn0 4 (19.0%) 4 (26.7%) pn1 17 (81.0%) 11 (73.3%) UICC stge I - - II 20 (95.2%) 14 (93.3%) III/IV 1 (4.8%) 1 (6.7%) Histologicl grde G1 4 (19.0%) 3 (20.0%) G2 5 (23.8%) 7 (46.7%) G3 12 (57.2%) 5 (33.3%) Residul tumor ctegory R0 12 (57.2%) 12 (80.0%) R1 9 (4.8%) 3 (20.0%) Vessel invsion Positive 15 (71.4%) 9 (60.0%) Negtive 6 (28.6%) 6 (40.0%) Neurl invsion Positive 17 (81.0%) 13 (86.7%) Negtive 4 (19.0%) 2 (13.3%) Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88) nd the GEM group (n=36). predicting the outcome of PDAC ptients, even when specimens contin bundnt contminted cells. Discussion There is incresing evidence showing the usefulness of immunohistochemicl nlysis of moleculr mrkers, including EGFR, for predicting the clinicl outcome of PDAC ptients (10,11,13,25,26). Immunohistochemicl nlysis is vlid method s it shows protein expression. However, the clinicl introduction of immunohistochemicl ssessment for predicting sensitivity to chemotherpeutic gents is still problemtic due to difficulties in quntittive mesurement (inter-observer vritions in interprettion) nd the lck of clibrted quntifiction techniques (27-29). In ddition, only 10-20% of ptients with PDAC re cndidtes for curtive resection (3). Therefore, the remining 80 90% of ptients with dvnced PDAC need cytopthologicl ssessment with EUS-FNA, or pncretic juice, to predict their sensitivity to chemotherpeutic gents for individulized chemotherpy. The present nlysis of EGFR mrna is quntittive (even considering the smll mount of specimens vilble, including cytologicl specimens). For these resons, quntittive mrna nlysis of genes ssocited with tumor sensitivity, or with resistnce to nti-tumor gents, could be preferred to immunohistochemicl nlysis in clinicl setting. Furthermore, we introduced the use of LCM to obtin trget cells from EUS-FNA cytologicl specimens (17). As result, we found tht EGFR mrna levels in microdissected neoplstic cells were esier to distinguish thn those in WCP, suggesting tht quntifiction of the expression levels of individul genes in microdissected neoplstic cells could be potent tool for

10 638 fujit et l: EGFR mrna expression in PDAC Tble VII. Univrite survivl nlysis of conventionl prognostic fctors nd EGFR mrna expression in the GEM group (n=36). Chrcteristics Number of Medin DFS P-vlue Medin OS P-vlue 5-yer survivl cses (months) (months) rte EGFR mrna expression b b High % Low % Age yers % <65 yers % Gender Mle % Femle % Rdiotherpy Yes % No % pt ctegory pt1/pt % pt3/pt % pn ctegory b b pn % pn % UICC stge I II % III/IV % Histologicl grde G1/G % G % Residul tumor ctegory b b R % R % Vessel invsion Positive % Negtive % Neurl invsion Positive % Negtive % Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88) nd the GEM group (n=36); b p<0.05. Tble VIII. Multivrite DFS nlysis (Cox regression model) of conventionl prognostic fctors nd EGFR mrna expression levels in the GEM group (n=36). Chrcteristics Reltive risk 95% Confidence intervl P-vlue pn sttus (pn1) Residul tumor ctegory (pr1) b High EGFR levels (>0.058) b Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88) nd the GEM group (n=36); b p<0.05.

11 INTERNATIONAL JOURNAL OF ONCOLOGY 38: , Tble IX. Multivrite OS nlysis (Cox regression model) of conventionl prognostic fctors nd EGFR mrna expression levels in the GEM group (n=36). Chrcteristics Reltive risk 95% Confidence intervl P-vlue pn sttus (pn1) b Residul tumor ctegory (pr1) b High EGFR levels (>0.058) b Cut-off vlue (0.058) ws determined by recursive descent prtition nlysis of ll ptients (n=88) nd the GEM group (n=36); b p<0.05. Figure 6. Quntittive nlyses of EGFR mrna in EUS-FNA cytologicl specimens. Representtive microgrphs of cytologicl specimens obtined from ptients with PDAC who underwent EUS-FNA cytologicl exmintion (A nd B). Most smples contined lrge mount of blood nd inflmmtory cells with scrce clusters of neoplstic cells. (C) Quntittive nlysis of EGFR in EUS-FNA cytologicl specimens (n=20). Although we did not detect cler chnges in expression levels in the WCP smples, we distinguished smples hving higher nd lower EGFR expression levels in the microdissected neoplstic cells (C). predicting sensitivity to nti-tumor gents, even when specimens contin contminted cells. However, further investigtions, including prospective studies, re required before this pproch cn be introduced into the clinicl setting. As EGFR plys crucil role in controlling the ctivity of the Rs/MAPK signling pthwy (8,9), gret efforts hve been mde to develop strtegies trgeting EGFR (30). In xenogrft models of pncretic cncer, the combintion of gemcitbine nd EGFR-trgeted therpy significntly inhibited lymph node nd liver metstses nd improved OS (16). A rndomized, plcebo-controlled phse III tril compring erlotinib, n EGFR tyrosine kinse inhibitor (TKI), plus gemcitbine to gemcitbine lone in ptients with loclly dvnced or metsttic pncretic cncer, demonstrted tht

12 640 fujit et l: EGFR mrna expression in PDAC the combintion of erlotinib nd gemcitbine provided smll, yet sttisticlly significnt survivl benefit (31). However, the efficcy of EGFR TKI in pncretic cncer trils hs not met expecttions, not s it hs in proportion of ptients with non-smll cell lung cncer (NSCLC) (30). This is likely due to differences in the presence of ctivting muttions within EGFR, which re ssocited with prolonged responses in NSCLC (32). Mny studies hve identified ctivting EGFR gene muttions in only smll number of cses in PDAC ptients (33,34). However, Tn et l demonstrted tht higher genetic mplifiction of the EGFR region of chromosome 7 is ssocited with better clinicl responses to erlotinib tretment in dvnced NSCLC ptients (35). Therefore, there is possibility tht quntittive nlysis of EGFR mrna expression levels could be helpful in predicting sensitivity to erlotinib in PDAC ptients. However, further investigtions incorporting lrger ptient numbers re required to evlute the usefulness of this pproch. In conclusion, we demonstrte tht quntittive nlysis of EGFR mrna expression using FFPE tissue smples is useful for predicting the prognosis of PDAC ptients receiving gemcitbine-bsed AC. In ddition, quntittive nlysis of EGFR mrna in neoplstic cells microdissected from EUS-FNA specimens is useful for determining tretment for ptients with PDAC, even when the tumor is unresectble. Thus, quntittive nlysis of genes ssocited with sensitivity to cytotoxic gents could be potent tool for individulized chemotherpy. Acknowledgements We would like to thnk Mr. F. Ohkubo (Deprtment of Pthology, Kyushu University Hospitl) for skillful cytologicl exmintion nd nlysis. We lso thnk Ms. S. Sdtomi, Ms. M. Sto, Ms M. Ohmori nd Ms. E. Mnbe (Deprtment of Surgery nd Oncology, Kyushu University Hospitl) for skillful exmintion nd nlysis. 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