Possible predictive role of cancer/testis antigens in breast ductal carcinoma in situ

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1 ONCOLOGY LETTS Possible predictive role of cncer/testis ntigens in brest ductl crcinom in situ ANA ROGULJIC 1, GULIO SPAGNOLI 2, ANTONIO JURETIC 3, BOZENA SARCEVIC 4, MARIJA BANOVIC 5 nd LIDIJA BEKETIC ORESKOVIC 6 1 Deprtment of Rdition nd Medicl Oncology, Sisters of Mercy University Hospitl Center, University Hospitl for Tumors, 00 Zgreb, Croti; 2 Deprtment of Biomedicine, University Hospitl Bsel, 4031 Bsel, Switzerlnd; 3 Deprtment of Oncology, Clinicl Hospitl Center Zgreb, University of Zgreb School of Medicine; 4 Deprtment of Oncology Pthology, University of Zgreb School of Medicine, Sisters of Mercy University Hospitl Center, University Hospitl for Tumors; 5 University of Zgreb School of Medicine; 6 Deprtment of Oncology, University of Zgreb School of Medicine, Sisters of Mercy University Hospitl Center, University Hospitl for Tumors, 00 Zgreb, Croti Received Mrch 5, 2018; Accepted September 27, 2018 DOI: /ol Abstrct. Cncer/testis ntigens (CTAs) re lrge fmily of tumor ssocited ntigens expressed in humn tumors of different histologicl origin, but not in norml tissues, with the exception of the testes nd plcent. Numerous immunohistochemicl studies hve reported ssocitions between CTA expression nd negtive estrogen receptor () sttus in brest tumors, nd demonstrted tht CTAs re frequently expressed in tumors with higher nucler grde. The expression of CTAs hs not been studied s extensively in ductl crcinom in situ (DCIS) s it hs been in invsive brest cncer. The present retrospective study included rchived prffin embedded specimens from 83 ptients dignosed with DCIS in the period between Jnury 2007 nd December The follow up time for locl recurrence rnged between 1 nd 8 yers (men, 5.02 yers). Antigens from the melnom ssocited ntigen gene (MAGE) fmily, nmely multi MAGE A, MAGE A1, MAGE A10 nd New York esophgel squmous cell crcinom 1 (NY ESO 1) ntigen, were evluted by immunostining nd their subcellulr loction ws investigted. Presence of tumor infiltrting lymphocytes (TILs) ws evluted on ll sections, together Correspondence to: Dr An Roguljic, Deprtment of Rdition nd Medicl Oncology, Sisters of Mercy University Hospitl Center, University Hospitl for Tumors, 197 Ilic, 00 Zgreb, Croti E mil: roguljic.n@gmil.com Abbrevitions: CTA, cncer/testis ntigen; DCIS, ductl crcinom in situ;, estrogen receptor; mab, monoclonl ntibody;, progesterone receptor; TILs, tumor infiltrting lymphocyte; TN, triple negtive Key words: ductl crcinom in situ, cncer testis ntigen, tumor infiltrting lymphocytes, melnom ssocited ntigen A10, New York esophgel squmous cell crcinom 1 ntigen with the histopthologicl vribles of DCIS. Specific tested ntigens exhibited ssocitions with histopthologicl prmeters for DCIS nd ll demonstrted sttisticlly significnt ssocitions with nucler stining, simultneous cytoplsmic nd nucler stining, nd locl recurrence. Antigen MAGE A10 demonstrted significnt ssocition with higher expression of (P=0.005) nd higher tumor nucler grde (P=0.001), cytoplsmic stining (P=0.029) nd ntigen NY ESO 1 with higher tumor size (P=0.001), expression of TILs (P=0.001) nd R1 resection (P=0.001). A χ 2 test reveled significnt ssocitions between simultneous cytoplsmic nd nucler stining nd locl recurrence (P=0.005), centrl necrosis (P=0.016), nd the expression of (P=0.003) nd progesterone receptor () (P=0.010). Additionl nlysis reveled n ssocition between ntigen MAGE A10 nd TILs (P=0.05). Additionl nlysis of TILs indicted tht they were significntly ssocited with tumor grde (P=0.023), centrl necrosis (P<0.001), (P=0.003) nd (P=0.029). Overll, CTAs from the MAGE fmily (MAGE A1, multi MAGE A nd MAGE A10) nd NY ESO 1 ssocite with histopthologicl predictive vribles of DCIS. The expression of ntigens NY ESO 1 nd MAGE A10 could serve n importnt role in the tretment of ptients with negtive histopthologicl predictive vribles, but further nlysis is required. Simultneous cytoplsmic nd nucler protein expression of MAGE A fmily nd NY ESO 1 CTAs my represent n independent mrker for locl recurrence. Tken together, the present dt suggest tht CTAs re not perfect indictors of invsiveness for DCIS, but could inform tretment strtegies for ptients when tken in combintion with other histopthologicl predictive vribles. However, this ws smll study nd further lrger studies will be necessry to confirm the current findings. Introduction Ductl crcinom in situ (DCIS) is non invsive type of brest cncer tht evolves in the milk ducts of the brest nd

2 2 ROGULJIC et l: POSSIBLE EDICTIVE ROLE OF CANC/TESTIS ANTIGENS IN BREAST DCIS remins locted there. DCIS is non obligte precursor of invsive brest cncer nd up to 40% of these lesions progress to invsive disese if untreted (1). The incidence of DCIS is rising, most likely due to incresed use of mmmogrphic screening nd the trnsition from screen film mmmogrphy to digitl mmmogrphy (2). DCIS is not one entity but heterogeneous group of t lest four subtypes (luminl A, luminl B, Her 2 overexpressed nd triple negtive very rre) (3). It remins uncler which type of DCIS is more likely to progress to invsive brest cncer nd therefore will require more intensive tretment. Cncer/testis ntigens (CTAs) re lrge fmily of tumor ssocited ntigens expressed in humn tumors of different histologicl origin, but not in norml tissues, with the exception of the testis nd plcent (4). This unique clss of tumor ssocited ntigens ws discovered in the erly 1990s nd the first to be identified ws melnom ssocited ntigen 1 (MAGE 1) in melnom ptients (5,6). CTAs my be divided into two lrge groups, depending on whether they re encoded on the X chromosome (X CTA genes) or not (non X CTA genes) (7). X CTA genes include the synovil srcom X (SSX) fmily, the GAGE/PAGE/XAGE super fmilies nd the MAGE A, MAGE C nd New York esophgel squmous cell crcinom 1 (NY ESO 1) multigene fmilies, mong others (7,8). Antigens in this group re widely nd vribly expressed mong tumors of different histotypes (4). Expression of CTAs is highly vrible nd my be observed frequently in melnoms nd bldder, lung, ovrin nd heptocellulr crcinoms, but rrely in renl, colon nd gstric cncer or hemtologicl mlignncies (9). In brest cncer, multiple immunohistochemicl studies hve reported n ssocition between CTA expression nd negtive estrogen receptor () sttus in brest tumors, nd hve demonstrted tht CTAs re frequently expressed in tumors with higher nucler grde (10,11). Spontneous humorl nd cell medited immune responses ginst severl CTAs, including MAGE A1 (6) nd NY ESO 1 ntigens (12) hs led to the proposl tht CTAs could represent ttrctive cncer immunotherpy trgets nd hs inspired reserch into the development of ntigen specific vccines (9). The expression of CTAs in DCIS hs not been studied s extensively s in invsive brest cncer. However, in two studies, the expression of CTAs in DCIS ws studied nd it ws demonstrted tht NY ESO1 is expressed in high proportion of DCIS tissues, prticulrly those tht re negtive (10,11). The present study investigted the expression of CTAs from the MAGE fmily (multi MAGE A, MAGE A1 nd MAGE A10) nd NY ESO 1 in DCIS, nd their ssocition with stndrd histopthologicl prmeters for DCIS [tumor size, tumor grde, expression of nd progesterone receptor (), necrosis nd mrgin] nd locl recurrence. The evlution of tumor infiltrting lymphocytes (TILs) ws lso performed. Mterils nd methods Ptients nd smples. This retrospective study included rchived prffin embedded specimens from 83 ptients dignosed with DCIS who underwent segmentectomy surgery t the University Hospitl for Tumors, Sisters of Mercy University Hospitl Center (Zgreb, Croti) between Jnury 2007 nd December The ptients were ll femle, ged between 40 nd 70 yers old (men ge 57.4 yers). All cses of surgiclly resected DCIS were reviewed in the current study, nd histopthologicl prmeters (tumor size, histologicl tumor grde, nd sttus, necrosis nd mrgin) were routinely ssessed nd recorded in dtbse. All ptients received rdiotherpy following brest conserving surgery (lumpectomy) nd certin ptients (those who were receptor positive) received hormone therpy for 5 yers. Follow up rnged between 1 nd 8 yers (men, 5.02 yers). This study received ethicl pprovl from the Sisters of Mercy University Hospitl Center. Written informed ptient consent ws received t the time of the mteril collection. Histology nd immunohistochemistry. Tumors were fixed in 10% buffered formlin for pproximtely 24 h t 4 C, cut t 3 4 milimeters nd smpled in 3 7 sections. The specimens were embedded in prffin, routinely cut nd stined with hemtoxylin nd eosin (H&E). In ech cse, the vilble H&E sections were reviewed nd slides with the deepest portion of tumor penetrtion were selected for immunohistochemicl nlysis. A totl of 4 new 5 µm sections were cut from the prffin embedded blocks of ech smple for nlysis. Tissue slides from prffin embedded brest cncer tumor smples were plced on Silne (3 minopropyltriethoxysilne, A 3,648, Sigm Aldruch, Merck KgA, Drmstdt, Germny). Following deprffiniztion, slides were heted in n 800 W microwve oven t mximum power for 8.5 min, held in 10 mmol/l citrte buffer (ph 6.0) for 5 min nd then rinsed with phosphte buffer solution (PBC, ph 7.2). Four monoclonl ntibodies were used to determine the expression of nlyzed proteins in DCIS (ntibodies re gift from Dr. Spgnoli, Bsel, Switzerlnd, they re not commercil ntibodies). Monoclonl ntibodies (mabs) recognizing the following CTAs were used: Anti MAGE A1 (clone 77B), Anti multi MAGE A (clone 57B), nti MAGE A10 (clone 3GA11) nd nti NY ESO 1 (clone D8.38). These mouse monoclonl ntibodies CTAs (77B, 57B, 3GA11 nd D8.38) mab were used undiluted (undiluted superntnts). 57B ws generted on immuniztion of mice with recombinnt MAGE A3 (13). However, this ntibody recognizes vriety of MAGE A molecules, nd it is considered multi MAGE A specific regent. D8.38 ntibody, recognizing NY ESO 1 nd its homologous LAGE 1 CTA, hs been previously described (14). 3GA11 ntibody recognizing MAGE A10 (15) nd 77B recognizing MAGE A1 hs lso been previously described (16). TMA stining ws performed s described previously (17). Briefly, tissue slides from prffin embedded brest cncer tumor smples were plced on Silne (3 minopropyltriethoxysilne, A 3648, Sigm, St. Louis MO, USA) nd incubted for 20 min in thermostt t 60 C. The sections were then deprffinized nd incubted for 3x5 min in 10 mmol/l of citrte buffer (ph 6.0) in microwve oven t 800 W. Subsequently, tissue slides were wshed with phosphte buffered sline (PBS) buffer (ph 7.2), nd endogenous peroxidse ctivity ws blocked by 5 min tretment with hydrogen peroxide (. S2023,

3 ONCOLOGY LETTS Tble I. Frequency nd percentge of study smples with given histopthologicl vribles. Prmeters n (%) P vlue Tumor size, mm < (60) > (40) < (22) 2, 3 65 (78) (41) 49 (59) TIL (54) 38 (46) Multi MAGE A < (16) 70 (84) MAGE A (51) 41 (49) MAGE A1 < (7) 77 (93) NY ESO 1 < (27) 61 (73) < (29) 59 (71) (37) 52 (63) R (47) 44 (53) Cytoplsmic stining (34) 55 (66) Nucler stining < (90) 8 (10) Cytoplsmic nd nucler stining < (82) 15 (18) Totl stining < (71) 24 (29) Locl recurrence < (92) 7 (8) Sttisticlly significnt result. TIL, tumor infiltrting lymphocyte; MAGE, melnom ssocited ntigen; NY ESO 1, New York esoph gel squmous cell crcinom 1 ntigen;, estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin. 3 Tble II. Sensitivity, specificity nd cutoff vlues for ntigen nd receptor detection. Prmeters Multi MAGE A MAGE A10 MAGE A1 NY ESO 1 Sensitivity, (%) Specificity, (%) Cutoff vlue, (%) >20 >30 >0 >0 >0 >0 MAGE, melnom ssocited ntigen; NY ESO 1, New York esoph gel squmous cell crcinom 1 ntigen;, estrogen receptor;, progesterone receptor. Figure 1. Immunohistochemistry stining of DCIS smples using mab specific to NY ESO 1 (clone D8.38) (observed in brown) nd MAGE A1 (mab clone 77B). Sections presented vrible (A) nucler NY ESO 1 stining, nd (B) cytoplsmic nd (C) nucler MAGE A1 stining. Originl mgnifiction, x10. DCIS, ductl crcinom in situ; NY ESO 1, New York esophgel squmous cell crcinom 1 ntigen; MAGE A1, melnom sso cited ntigen A1; mab, monoclonl ntibody.

4 4 ROGULJIC et l: POSSIBLE EDICTIVE ROLE OF CANC/TESTIS ANTIGENS IN BREAST DCIS Tble III. Mnn Whitney U test results of the expression of cncer/testis ntigens nd histopthologicl vribles. Prmeters Multi MAGE A MAGE A10 MAGE A1 NY ESO 1 Tumor size U 1,909 3,112 1,618 2,282 P vlue U 3,237 2,448 2,946 3,278 P vlue U 2,573 3,112 2,282 2,946 P vlue TILs U 2,116 3,320 1,826 2,490 P vlue U 2,988 2,697 2,697 3,361 P vlue U 2,697 2,988 2,407 3,071 P vlue R1 U 2,365 3,320 2,075 2,739 P vlue Cytoplsmic stining U 2,822 2,863 2,531 3,195 P vlue Nucler stining U 871 2, ,245 P vlue Cytoplsmic nd nucler stining U 1,162 2, ,535 P vlue Totl stining U 1,535 2,739 1,245 1,909 P vlue Locl recurrence U 830 2, ,203 P vlue Sttisticlly significnt result. U, Mnn Whitney U vlue; MAGE, melnom ssocited ntigen; NY ESO 1, New York esophgel squmous cell crcinom 1 ntigen; TIL, tumor infiltrting lymphocyte;, estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin. Dko, Crpinteri, CA, USA). Slides were then wshed with PBS buffer nd incubted for 90 min with multi MAGE A 57B, MAGE A1 77B, MAGE A10 3GA11 or NY ESO 1 D8.38 undiluted superntnts t room temperture. After wshing in PBS, bound primry ntibodies were detected using biotinylted nti mouse secondry ntibody (EnVision FLEX, High ph Kit, ctlogue number 8010; Dko, redy for use) for 45 min nd visulized with diminobenzidine s chromogen on Autostiner Link 48 (Dko). Slides were counterstined with hemtoxilyn, dehydrted, clered nd cover slipped. Melnoms nd testiculr tissues expressing CTAs from University Hospitl for Tumors, Sisters of Mercy University Hospitl Center (Zgreb, Croti) were used s positive controls throughout the study, nd helthy skin tissue nd unstined tumor cells served s the negtive control. The specimens were described s positive or negtive for TIL ccording to their presence in the smples. The positive cells were scored in whole tumor t x200 mgnifiction using light microscope on selected slides. All smples were exmined independently by three observers nd ny difference ws resolved by joint review.

5 ONCOLOGY LETTS 5 Tble IV. Multivrite logistic regression for cncer/testis ntigens nd histopthologicl prmeters. Prmeters Multi MAGE A MAGE A10 MAGE A1 NY ESO 1 Cytoplsmic stining P vlue OR % CI Nucler stining P vlue OR 25.2x x % CI Cytoplsmic nd nucler stining P vlue OR 28.3x x % CI Totl stining P vlue OR % CI Tumor size P vlue OR % CI P vlue OR % CI P vlue OR % CI TILs P vlue OR % CI P vlue OR % CI P vlue OR % CI R1 P vlue OR % CI Locl recurrence P vlue OR 38.4x % CI x x10 48 Sttisticlly significnt result. MAGE, melnom ssocited ntigen; NY ESO 1, New York esophgel squmous cell crcinom 1 ntigen; OR, odds rtio; TIL, tumor infiltrting lymphocyte;, estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin.

6 6 ROGULJIC et l: POSSIBLE EDICTIVE ROLE OF CANC/TESTIS ANTIGENS IN BREAST DCIS Tble V. Associtions between histopthologicl vribles nd stining (χ 2 test). Cytoplsmic Nucler Nucler nd Totl Prmeters R1 stining stining cytoplsmic stining stining Tumor size < TILs R Locl recurrence Sttisticlly significnt result., estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin; TIL, tumor infiltrting lymphocyte. Scoring. Multi MAGE A, MAGE A1, MAGE A10 nd NY ESO 1 stining results were scored using the Allred scoring system (18). This method tkes into ccount percentges of positive cells (scored on 0 3 scle) nd the intensity of their stining (scored on 0 3 scle). If the expression of CTAs ws detectble in <10% of tumor cells it ws scored s 1, in 10 50% of tumor cells it ws scored s 2, or in >50% of tumor cells it ws scored s 3. Score 0 ws ttributed to negtive smples. The percentge of positive cells ws then multiplied by the intensity of stining, 0, no rection; 1, wek rection; 2, moderte rection; 3, strong rection nd the finl score rnged between 0 (no stining) nd 9 (diffuse nd strong stining). The finl results were further clssified s 0 (no stining), 1 (score 1, 2 or 3), 2 (score 4, 5 or 6) nd 3 (score 7, 8 or 9). Stining ws considered positive (score 2 or 3) where ll or mjority of the tumor cells were stined. Sttisticl nlysis. Dt were nlyzed using Microsoft Excel 2010 (Microsoft Corportion, Redmond, WA, USA), MedClc (version ; MedClc Softwre bvb, Ostend, Belgium) nd IBM SPSS Sttistics (version 21.0; IBM Corp., Armonk, NY, USA). Descriptive sttistics were clculted for the expression of proteins from the MAGE fmily (multi MAGE A, MAGE A1 nd MAGE A10), NY ESO 1, stndrd histopthologicl prmeters for DCIS (tumor size, tumor grde, expression of nd, necrosis, mrgin nd TILs) nd locl recurrence. Cutoffs were estblished by receiver operting chrcteristic curve nlysis. Associtions between vribles were nlyzed with the Mnn Whitney U signed rnk test nd with Fisher's exct test. The χ 2 test ws used for the nlysis of ssocitions between histopthologicl vribles, stining nd TILs. Furthermore, multivrite logistic regression model ws used to predict the effect of series of vribles (ntigens) on binry response vrible (histopthologicl prmeters for DCIS). P<0.05 ws considered to indicte sttisticlly significnt difference. Results Ptient popultion. This retrospective study included totl of 83 ptients who were dignosed with DCIS nd underwent surgery between Jnury 2007 nd December Dt on tumor size, tumor grde, expression of nd, necrosis, mrgin, TILs, nd expression of CTAs multi MAGE A, MAGE A1, MAGE A10 nd NY ESO 1 in smples re summrized in Tble I. Antigens MAGE A1, multi MAGE A, NY ESO 1 nd MAGE A10 were expressed in 93, 84, 73 nd 49% of cses, respectively (Tble I). The cutoff vlues for the detection of, nd the CTAs were clculted (Tble II). Since expression of CTAs hs been previously detected in different intrcellulr loctions (19), the present study focused on three different stining ptterns, nmely nucler, cytoplsmic, nd simultneous cytoplsmic nd nucler expression of multi MAGE A, MAGE A1, MAGE A10 nd NY ESO 1 in brest DCIS cells (Fig. 1). This specifies wht ws focused on, but not wht ws found. Associtions between multi MAGE A, MAGE A1, MAGE A10 nd NY ESO 1 expression nd histopthologicl prmeters of DCIS. All the tested ntigens exhibited ssocitions with histopthologicl prmeters for DCIS, nd they ll demonstrted sttisticlly significnt ssocitions with nucler stining, simultneous cytoplsmic nd nucler stining, nd locl recurrence (Tble III). Antigen MAGE A10 ws significntly ssocited with higher expression of (P=0.005), higher tumor grde (P=0.001) nd cytoplsmic stining (P=0.029), nd ntigen NY ESO 1 with lrger tumor size (P=0.001), expression of TILs (P=0.001) nd R1 resection (P=0.001). The multivrite logistic regression model, ntigen MAGE A1 expression demonstrted significnt ssocition with cytoplsmic stining (P=0.006, Tble IV). The ssocition between the subcellulr expression pttern of CTA nd histopthologicl prmeters ws nlyzed. The χ 2 test identified significnt ssocition between the cytoplsmic stining pttern nd the expression of (P=0.012) nd (P=0.029). It lso identified significnt ssocition between locl recurrence nd cytoplsmic nd nucler CTA expression pttern in brest DCIS cells (P=0.005). Simultneous cytoplsmic nd nucler stining ws lso significntly ssocited with centrl necrosis (P=0.016), nd the expression of (P=0.003) nd (P=0.010) (Tble V). The nlysis of ssocition between cytoplsmic stining, nucler stining, cytoplsmic nd nucler stining, nd totl stining with histopthologicl vribles showed similr results (Tble VI).

7 ONCOLOGY LETTS 7 Tble VI. Cytoplsmic stining, nucler stining, cytoplsmic nd nucler stining, nd totl stining in ssocition with histopthologicl vribles. Cytoplsmic stining Nucler stining Cytoplsmic nd nucler stining Totl stining Negtive, Positive, Negtive, Positive, Negtive, Positive, Negtive, Positive, Prmeters n (%) n (%) n (%) P vlue n (%) n (%) P vlue n (%) n (%) P vlue n (%) n (%) P vlue Tumor size, mm < (60) 14 (28) 36 (72) (94) 3 (6) (84) 8 (16) (72) 14 (28) > (40) 14 (42) 19 (58) 28 (85) 5 (15) 26 (79) 7 (21) 23 (70) 10 (30) 1 18 (22) 6 (33) 12 (67) (83) 3 (17) (94) 1 (6) (78) 4 (22) , 3 65 (78) 22 (34) 43 (66) 60 (92) 5 (8) 51 (78) 14 (22) 45 (69) 20 (31) 34 (41) 8 (24) 26 (76) (91) 3 (9) (94) 2 (6) (79) 7 (21) (59) 20 (41) 29 (59) 44 (90) 5 (10) 36 (74) 13 (26) 32 (65) 17 (35) TILs 45 (54) 12 (27) 33 (73) (89) 5 (11) (89) 5 (11) (73) 12 (27) (46) 16 (42) 22 (58) 35 (92) 3 (8) 28 (74) 10 (26) 26 (68) 12 (32) Multi MAGE A 13 (16) 5 (38) 8 (62) () 0 (0) () 0 (0) (82) 7 (18) (84) 23 (33) 47 (67) 62 (89) 8 (11) 55 (79) 15 (21) 27 (61) 17 (39) MAGE A10 42 (51) 13 (31) 19 (69) (93) 3 (7) (88) 5 (12) (79) 9 (21) (49) 15 (37) 26 (63) 36 (88) 5 (12) 31 (76) 10 (24) 26 (63) 15 (37) MAGE A1 6 (7) 5 (83) 1 (17) () 0 (0) () 0 (0) (50) 3 (50) (93) 23 (30) 54 (70) 69 (90) 8 (10) 62 (80) 15 (20) 56 (73) 21 (27) NY ESO 1 22 (27) 7 (32) 15 (68) () 0 (0) (91) 2 (9) (77) 5 (23) (73) 21 (34) 40 (66) 53 (87) 8 (13) 48 (79) 13 (21) 42 (69) 19 (31) 24 (29) 13 (54) 11 (46) (92) 2 (8) (62) 9 (38) (50) 12 (50) (71) 15 (25) 44 (75) 53 (90) 6 (10) 53 (90) 6 (10) 47 (80) 12 (20) 31 (37) 15 (48) 16 (52) (93) 2 (7) (68) 10 (32) (55) 14 (45) (63) 13 (25) 39 (75) 46 (88) 6 (12) 47 (90) 5 (10) 42 (81) 10 (19)

8 8 ROGULJIC et l: POSSIBLE EDICTIVE ROLE OF CANC/TESTIS ANTIGENS IN BREAST DCIS Tble VI. Continued. Cytoplsmic stining Nucler stining Cytoplsmic nd nucler stining Totl stining Negtive, Positive, Negtive, Positive, Negtive, Positive, Negtive, Positive, Prmeters n (%) n (%) n (%) P vlue n (%) n (%) P vlue n (%) n (%) P vlue n (%) n (%) P vlue R1 39 (47) 9 (23) 30 (77) (92) 3 (8) (90) 4 (10) (82) 7 (18) (53) 19 (43) 25 (57) 39 (89) 5 (11) 33 (75) 11 (25) 27 (61) 17 (39) Locl recurrence 76 (92) 24 (32) 52(68) (89) 8 (11) (85) 11 (15) (74) 20 (26) (8) 4 (57) 3 (43) 7 () 0 (0) 3 (43) 4 (57) 3 (43) 4 (57) Sttisticlly significnt result. TIL, tumor infiltrting lymphocyte; MAGE, melnom ssocited ntigen; NY ESO 1, New York esophgel squmous cell crcinom 1 ntigen;, estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin. Additionl nlysis of the ssocition between the expression of CTA nd stndrd histopthologicl prmeters with Fisher's exct test indicted tht expression of MAGE A10 ntigen ws ssocited with TILs (P=0.05; Tble VII). The dditionl nlysis of TILs nd histopthologicl vribles indicted significnt ssocition with tumor grde (P=0.023), centrl necrosis (P<0.001) nd expression of (P=0.003) nd (P=0.029) (Tble VIII). Multivrite logistic regression models indicted n ssocition between TILs nd tumor size (P=0.038), tumor grde (P=0.030), centrl necrosis (P=0.001), nd expression of (P=0.005) nd (P=0.031) (Tble IX). Discussion The role of CTAs hs not been studied s extensively in DCIS s it hs in invsive brest cncer. A smll number of studies hve nlyzed the expression of CTAs in DCIS, but none hve nlyzed the ssocitions between the expression of CTAs nd histopthologicl predictive vribles. In the current study, ssocitions between histopthologicl predictive vribles nd the expression of CTAs from the MAGE fmily (multi MAGE A, MAGE A1 nd MAGE A10) nd NY ESO 1 were evluted. Cbllero et l (10) recently reported tht ntigen NY ESO 1 is predictor of good prognosis in ptients with DCIS. In this study, NY ESO 1 ws predominntly expressed in negtive DCIS nd ptients who expressed NY ESO 1 ntigen did not suffer from recurrence over 10 yer period. Therefore, it ws concluded tht NY ESO 1 hs protective effect nd is expressed in ptients who will not subsequently develop invsive brest cncer. In the present study, ll exmined ntigens were demonstrted to be ssocited with histopthologicl predictive vribles of DCIS. Different stining ptterns (cytoplsmic, nucler, or cytoplsmic nd nucler) nd nucler protein expression of MAGE A fmily nd NY ESO 1 CTAs were observed. All tested ntigens were significntly ssocited with nucler stining, simultneous cytoplsmic nd nucler stining, nd locl recurrence. Using the multivrite logistic regression model, ntigen MAGE A1 expression demonstrted significnt ssocition with cytoplsmic stining (P=0.006). A similr stining pttern for MAGE A ntigen hs been reported previously in DCIS nd in invsive brest cncer, s well s in other mlignnt tumors (9,12,20). tbly, simultneous cytoplsmic nd nucler stining ws significntly ssocited with locl recurrence, centrl necrosis, nd the expression of nd. This ws lso previously observed in hed nd neck crcinom, where simultneous cytoplsmic nd nucler protein expression of MAGE A fmily nd NY ESO 1 CTAs represented n independent mrker for poor survivl (19). In the present study, ntigen MAGE A10 ws reveled to be significntly ssocited with higher expression of nd higher tumor grde, nd ntigen NY ESO 1 with higher tumor size, expression of TILs nd R1 resection. Antigen NY ESO 1 ws predominntly expressed in negtive DCIS, which is consistent with the results from previous study (10). An ssocition between the expression of MAGE A10 nd TILs ws lso observed (P=0.05). In the current nlysis of TILs nd their significnce in DCIS, significnt ssocition ws identified between TILs nd tumor grde, centrl

9 ONCOLOGY LETTS 9 Tble VII. Fisher's exct test results of the expression of cncer/testis ntigens ginst histopthologicl vribles. Multi MAGE A MAGE A10 MAGE A1 NY ESO 1 Negtive, Positive, Negtive, Positive, Negtive, Positive, Negtive, Positive, Prmeters n (%) n (%) n (%) P vlue n (%) n (%) P vlue n (%) n (%) P vlue n (%) n (%) P vlue Tumor size, mm < (60) 10 (20) 40 (80) (50) 25 (50) (8) 46 (92) (28) 36 (72) > (40) 3 (9) 30 (91) 17 (51) 16 (49) 2 (6) 31 (94) 8 (24) 25 (76) 1 18 (22) 4 (22) 14 (78) (61) 7 (39) (11) 16 (89) (17) 15 (83) , 3 65 (78) 9 (14) 56 (86) 31 (48) 34 (52) 4 (6) 61 (94) 19 (29) 46 (71) 34 (41) 8 (23) 26 (77) (62) 13 (68) (12) 30 (88) (26) 25 (74) (59) 5 (10) 44 (90) 21 (43) 28 (57) 2 (4) 47 (96) 13 (26) 36 (74) TILs 45 (54) 10 (22) 35 (78) (60) 18 (40) (7) 42 (93) (31) 31 (69) (46) 3 (8) 35 (92) 15 (40) 23 (60) 3 (8) 35 (92) 8 (21) 30 (79) 24 (29) 5 (21) 19 (79) (37) 15 (63) 0. 2 (8) 22 (92) (29) 17 (71) (71) 8 (14) 51 (86) 33 (56) 26 (44) 4 (7) 55 (93) 15 (25) 44 (75) 31 (37) 7 (23) 24 (77) (42) 18 (58) (10) 28 (90) (29) 22 (71) (63) 6 (11) 46 (89) 29 (56) 23 (44) 3 (6) 49 (94) 13 (25) 39 (75) R1 39 (47) 7 (18) 32 (82) (54) 18 (46) (8) 36 (92) (23) 30 (77) (53) 6 (14) 38 (86) 21 (48) 23 (52) 3 (7) 41 (93) 13 (29) 31 (71) Locl recurrence 76 (92) 13 (17) 63 (83) (51) 37 (49) (8) 70 (92) (29) 54 (71) (8) 0 (0) 7 () 3 (43) 4 (57) 0 (0) 7 () 0 (0) 7 () MAGE, melnom ssocited ntigen; NY ESO 1, New York esophgel squmous cell crcinom 1 ntigen; TIL, tumor infiltrting lymphocyte;, estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin.

10 10 ROGULJIC et l: POSSIBLE EDICTIVE ROLE OF CANC/TESTIS ANTIGENS IN BREAST DCIS Tble VIII. Anlysis of ssocitions between tumor infiltrting lymphocytes nd histopthologicl vribles (χ 2 test). Prmeters P vlue Tumor size < R Locl recurrence Sttisticlly significnt result., estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin. Acknowledgements The present study ws presented t the 25th Biennil Congress of the Europen Assocition for Cncer Reserch, June 30 July , Amsterdm, the Netherlnds nd published s bstrct no. PO 341 in ESMO Open Journl 3 (Suppl 2), Funding funding ws received. Avilbility of dt nd mterils The dtsets used nd/or nlyzed during the current study re vilble from the corresponding uthor on resonble request. Authors' contributions Tble IX. Multivrite logistic regression nlysis of tumor infiltrting lymphocytes ginst histopthologicl vribles. Prmeters P vlue OR 95% CI Tumor size R Locl recurrence Sttisticlly significnt result. OR, odds rtio; CI, confidence intervl;, estrogen receptor;, progesterone receptor; R1, positive surgicl mrgin. necrosis, nd negtive nd sttus. These findings re consistent with previous studies (21 23). In summry, ssocitions between CTAs from the MAGE fmily (MAGE A1, multi MAGE A nd MAGE A10) nd NY ESO 1, nd histopthologicl predictive vribles of DCIS, were reveled. An ssocition ws lso observed between the MAGE A10 ntigen nd the presence of TILs. These results indicte tht MAGE A10 nd NY ESO 1 my serve function in DCIS nd could present potentil trget for novel tretment strtegy. Additionl nlysis in lrger group of ptients will be required to evlute this further. Simultneous cytoplsmic nd nucler protein expression of MAGE A fmily nd NY ESO 1 CTAs my represent n independent mrker for locl recurrence. In conclusion, CTAs re not perfect indictors of invsiveness for DCIS, but in combintion with other histopthologicl predictive vribles, they could inform tretment strtegies for ptients. However, the present study ws smll nd fresh frozen tissue smples were not vilble, therefore the dditionl nlysis on mrna nd protein level ws not performed. Further lrger studies re wrrnted to expnd the cohort of ptients under investigtion nd further support the present dt t the gene expression level. AR, GS nd AJ nlyzed nd interpreted the ptient dt regrding the expression of CTAs from the MAGE fmily (multi MAGE A, MAGE A1 nd MAGE A10) nd NY ESO 1 in DCIS, nd their ssocition with stndrd histopthologicl prmeters for DCIS [tumor size, tumor grde, expression of nd progesterone receptor, necrosis nd mrgin. nd locl recurrence. BS performed the histologicl exmintion of the smples. MB contributed in sttisticl nlysis of dt. LBO red nd revised the rticle, nd contributed to dt interprettion nd conception of the present study. All uthors red nd pproved the finl mnuscript. Ethics pprovl nd consent to prticipte The present study received ethicl pprovl from the Sisters of Mercy University Hospitl Center. Written informed ptient consent ws received. Ptient consent for publiction Ptients provided their consent for the publiction of ny dt/ssocited imges. Competing interests The uthors declre tht they hve no competing interests. References 1. Lopez Grci MA, Geyer FC, Lcroix Triki M, Mrchió C nd Reis Filho JS: mbrest cncer precursors revisited: Moleculr fetures nd progression pthwys. Histopthology 57: , Virnig BA, Tuttle TM, Shmliyn T nd Kne RL: Ductl crcinom in situ of the brest: A systemtic review of incidence, tretment, nd outcomes. J Ntl Cncer Inst 102: , Clrk SE, Wrwick J, Crpenter R, Bowen RL, Duffv SW nd Jones JL: Moleculr subtyping of DCIS: Heterogeneity of brest cncer reflected in pre invsive disese. Br J Cncer 104: , Frtt E, Corl S, Covre A, Prisi G, Colizzi F, Dnielli R, Nicoly HJ, Siglotti L nd Mio M: The biology of cncer testis ntigens: Puttive function, regultion nd therpeutic potentil. Mol Oncol 5: , 2011.

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