Journal of Breast Cancer

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1 Journal of Breast Cancer ORIGINAL ARTICLE J Breast Cancer 216 December; 19(4): Exression of T-Lymhocyte Markers in Human Eidermal Growth Factor Recetor 2-Positive Breast Cancer Changro Lee 1, Seho Park 1,2, Joo Heung Kim 1, Sung Mook Lim 1, Hyung Seok Park 1, Seung Il Kim 1, Byeong-Woo Park 1 1 Deartment of Surgery, Yonsei University College of Medicine, Seoul; 2 Frontier Research Institute of Convergence Sorts Science, Yonsei University, Seoul, Korea Purose: The resent study aimed to examine the clinical imlications of CD4, CD8, and FOXP3 exression on the rognosis of human eidermal growth factor recetor 2 (HER2)-ositive breast cancer using a web-based database, and to comare the immunohistochemical exression of T-lymhocyte markers using rimary and metastatic HER2-ositive tumor tissues before and after HER2-targeted theray. Methods: Using the cbioportal for Cancer Genomics and Kalan-Meier lotter, the mrna exression, association between T-lymhocyte markers, and survival in HER2-ositive cancers were investigated according to various cutoff levels. Immunohistochemistry analysis was erformed using aired rimary and metastatic tissues of 29 HER2- ositive tumors treated with systemic chemotheray and HER2- directed theray. Results: HER2 mrna was mutually exclusive of T-lymhocyte markers, and a significant correlation between T- cell markers was observed in the cbioportal for Cancer Genomics. According to analysis of the Kalan-Meier lotter, the imact of T-lymhocyte marker exression on survival was statistically insignificant in clinical HER2-ositive tumors, irresective of the cutoff levels. However, in the intrinsic HER2-ositive subtye, the individual analyses of T-cell markers excet for FOXP3 and combined analysis showed significantly favorable survival irresective of cutoff oints. Although the small clinical samle size made it difficult to show the statistical relevance of immunohistochemistry findings, good resonses to neoadjuvant treatments might be associated with ositive exression of combined T-lymhocyte markers, and aroximately half of the samles showed discordance of combined markers between baseline and resistant tumors. Conclusion: T-lymhocyte markers could be favorable rognostic factors in HER2-ositive breast cancers; however, a consensus on atient section criteria, detection methods, and cutoff value could not be reached. The resistance to HER2-directed theray might involve different and ersonalized mechanisms, and further research is required to understand the association between immune function and HER2 exression and to overcome the resistance mechanisms to HER2-targeted theraies. Key Words: Biomarkers, Breast neolasms, Drug resistance, Human ERBB2 rotein, T-lymhocytes INTRODUCTION Aroximately 2% to 3% of breast cancers are human eidermal growth factor recetor 2 (HER2) overexressing or amlifying carcinomas, and these tumors were associated with oor survival before the era of targeted theray [1]. Incororation of trastuzumab into systemic chemotheray has Corresondence to: Seho Park Deartment of Surgery, Yonsei University College of Medicine, 5-1 Yonsei-ro, Seodaemun-gu, Seoul 3722, Korea Tel: , Fax: sh125@yuhs.ac This research was suorted by Korea Breast Cancer Foundation (Grant number: ) and the Ministry of Education of the Reublic of Korea and the National Research Foundation of Korea (NRF- 215S1A5B836349). Received: July 26, 216 Acceted: Setember 17, 216 shown imroved survival of atients with these aggressive tumors in the metastatic and adjuvant setting [2,3]. However, certain HER2-ositive cancers eventually develo resistance to theray and large number of recurrent or metastatic tumors are still incurable desite the existence of newly develoed HER2-targeted theraies, such as laatinib, ertuzumab, and trastuzumab emtansine [4]. Evading immune destruction is one of the hallmarks of cancer and immunity related molecules can be targets for anticancer drugs [5]. One mechanism through which trastuzumab exerts its function is antibody-deendent cellular cytotoxicity (ADCC), and therefore, evasion from ADCC has been suggested as a ossible resistance mechanism to targeted theray [6,7]. It has been reorted that anti-her2 CD4+ Th1 resonse, CD8+/FOXP3+ tumor infiltrating lymhocyte (TIL) ratio, or tumor-associated lymhocytes can be useful 216 Korean Breast Cancer Society. All rights reserved. ISSN This is an Oen Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (htt://creativecommons.org/ eissn licenses/by-nc/4.) which ermits unrestricted non-commercial use, distribution, and reroduction in any medium, rovided the original work is roerly cited.

2 386 Changro Lee, et al. redictors of the athological resonse to neoadjuvant chemotheray [8-1]. Data from clinical trials suggested that atients with CD8+ T cells or increased number of TILs might be associated with better survival when trastuzumab was administered [11,12]. However, methodological issues concerning the evaluation of TILs have been debated [13]. Recently, an international TILs Working Grou ublished recommendations for the evaluation of TILs in breast cancer; however, a formal recommendation for a clinically relevant TIL threshold has not yet been suggested [14]. This study aimed to exlore the exression status and the association between T-lymhocyte markers as well as to investigate the rognostic role of CD4, CD8, and forkhead P3 (FOXP3) exression in HER2-ositive breast cancer using a web-based database. Clinically clear cutoff values for T-cell marker exression have not been established and various cutoff levels were included in the web-based analysis. In addition, we comared the immunohistochemical exressions of CD4, CD8, and FOXP3 before and after HER2-targeted theray in rimary or metastatic HER2-ositive breast cancer atients to better understand the clinical imlications of T-cell markers on the mechanisms of resistance to targeted theray. METHODS Web-based bioinformatics analysis Genomic analysis was erformed for exloring the associations between HER2 and T-lymhocytes markers of CD4, CD8, and FOXP3 through the cbioportal for Cancer Genomics (htt:// which rovides web-based visualization, analysis and download of large-scale cancer genomic datasets including The Cancer Genome Atlas (TCGA) data [15,16]. We used the Breast Invasive Carcinoma (TCGA, Provisional) dataset, which includes mrna exression and clinicoathological information of 1,15 samles obtained from 1,98 atients in May 216. The RNA Seq Version 2 (RNA-Seq by Exectation Maximization [RSEM]) for each gene was selected to generate an OncoPrint in the cbioportal for visualizing the genetic alteration and to investigate the mutual exclusivity or co-occurrence among alterations in biomarkers of whole analyzed samles. We downloaded the mrna exression data of CD4, CD8 and FOXP3 and clinical HER2 information data to exlore the associations between T-lymhocyte markers in HER2-ositive breast cancers. Using variables of immunohistochemistry (IHC) HER2 and HER2 fluorescence in situ hybridization (FISH) status in the TCGA dataset as variables, IHC-HER2 ositive cases, HER2 FISH status ositive cases, or IHC-HER2 equivocal cases with HER2 FISH-ositivity were considered to have HER2-ositive tumors. The robability of relase-free survival according to biomarkers was calculated using the Kalan-Meier lotter database (htt://kmlot.com/analysis) [17]. It is an online tool that allows analysis of the effects of 54,675 genes on survival by using 1,188 cancer samles including 4,142 breast cancer atients with a mean follow-u duration of 69 months in May 216. Survival and gene exression data were derived from the Gene Exression Omnibus (Affymetrix microarray only), Euroean Genome-henome Atlas, and TCGA. The Affymetrix robe set IDs selected for the evaluation of CD4, CD8, and FOXP3 exressions were 23547_at, 25758_at, and _ at, resectively, in the resent study. To analyze the rognostic value of a articular gene, the atient samles were slit into two grous according to various quantile exressions of the roosed biomarker. Multile genes were also analyzed through a multigene classifier using the mean exression levels of selected biomarkers. Hazard ratio with 95% confidence interval and log-rank -value were calculated and dislayed on the webage. Patient selection Using tissue blocks reviously archived from HER2-ositive breast cancer atients who received systemic chemotheray combined with trastuzumab or tyrosine kinase inhibitors at the Severance Hosital of Yonsei University College of Medicine, Seoul, Reublic of Korea, between January 27 and December 211, the exressions of CD4, CD8, and FOXP3 on IHC were investigated to comare the T-lymhocytes markers before and after targeted theray [18]. Briefly, tumor samles before and after HER2-directed theraies were available in most cases; however, five out of 29 atients received targeted theray in the neoadjuvant setting and achieved athologic comlete resonse (CR) after curative surgery. Therefore, tissue blocks after targeted theray could not be obtained and the five atients with CR were defined as good resonders in the resent study. Of 29 atients, 24 eventually exerienced disease rogression or recurrence desite the administration of targeted theray and were defined as oor resonders in the study. The baseline exression of T-lymhocyte markers between five good and 24 oor resonders were comared. Among the 24 oor resonders, the T-cell markers of six recurrent or metastatic tumor samles could not be evaluated. Thus, using 18-matched baseline and resistant tumor samles before and after targeted theray, the exression of T-lymhocyte markers was comared. In total, 47 tissue blocks from 29 atients were analyzed. The resent study was aroved by the Institutional Review Board of Severance Hosital, Yonsei University Health System, Seoul, Reublic of Korea (IRB number: 4-

3 T-lymhocyte Markers in HER2-Positive Breast Cancer ). The requirement for written informed consent was waived. Clinicoathological arameters and immunohistochemical staining Clinicoathological information including treatment modalities or exression of hormone recetors and HER2 was obtained from the review of medical records and athology reorts. Chemotheraeutic agents of 29 atients were aclitaxel in 14 atients, anthracycline lus cyclohoshamide followed by taxane in 11, anthracycline lus taxane in two, and anthracycline lus cyclohoshamide in one. One atient was treated with cyclohoshamide, methotrexate, and 5-fluorouracil due to intolerability after first administration of anthracycline lus cyclohoshamide. Tumors with 1% nuclear-stained cells by IHC were considered ositive for estrogen recetor (ER) and rogesterone recetor according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/ CAP) guidelines [19]. The HER2 stains were scored as, 1+, 2+, or 3+ according to ASCO/CAP guidelines [2]. In cases with a HER2 2+ result, FISH was erformed using a PathVysion HER2 DNA Probe Kit (Vysis, Downers Grove, USA) and HER2 gene amlification was defined as a HER2 gene/chromosome 17 coy number ratio 2. according to ASCO/CAP guidelines [2]. HER2 was considered ositive in cases with an IHC score of 3+ or gene amlification by FISH. After reviewing the archival hematoxylin and eosin stained slides, IHC was erformed using the whole sections of formalin-fixed, araffin-embedded tissue blocks. Briefly, 3-µm thick whole sections were dearaffinized and rehydrated in usual manner. Antigen retrieval was erformed using an electronic ressure cooker for 1 minutes in Trilogy buffer (Cell Marque Co., Rocklin, USA). After treatment with hydrogen eroxide block solution for 1 minutes, background staining was blocked by incubation in Ultra V Block solution (Thermo Scientific/ Lab Vision, Fremont, USA) for 5 minutes at room temerature. After incubation with rimary antibodies against CD4 (diluted 1:1, M3352; Sring Bioscience Cor., Pleasanton, USA), CD8 (diluted 1:1, M3162; Sring Bioscience Cor.), and FOXP3 (diluted 1:5, AB234; Abcam, Cambridge, UK), immunodetection was carried out using the UltraVision LP detection system (Thermo Scientific/Lab Vision) according to the manufacturer s instructions. Color was develoed with 3,3 -diaminobenzidine and slides were counterstained with Harris hematoxylin. The rimary antibody incubation ste was omitted in the negative control. Interretation of the immunohistochemical staining was erformed blindly to obtain clinical data of the atients. Arbitrary scoring rules of CD4 and CD8 lymhocytes were as follows: 1+, % to 33% staining; 2+, 34% to 66% staining; and 3+, 67% to 1% staining. FOXP3 was scored by counting the number of ositively stained cells, which was exressed as a ercentage of total lymhocytes. The cutoff value for FOXP3 ositivity was 1% of the stained cells. Statistical analysis Web-based bioinformatics statistics was automatically calculated by each website and the results were dislayed on the webage. The downloaded TCGA dataset was analyzed using correlation coefficient to exlore the association between T- lymhocyte markers in HER2-ositive tumors. Differences between the discrete variables in atients of Severance Hosital were evaluated by using the Fisher exact test. For the comarison of the means in the case of continuous numerical data, an indeendent two-samle t-test was used. A -value <.5 was considered statistically significant. All statistical analyses were erformed using SPSS version 23. (IBM Cor., Armonk, USA). RESULTS We first exlored the mrna exression status using the TCGA Provisional dataset, and information about the biomarker exression was obtained for 1,1 samles from 1,93 atients. Genetic alteration of CD4, CD8, FOXP3, and HER2 including mrna uregulation and downregulation was noted in 4%, 4%, 4%, and 16% of whole samles, resectively (Sulementary Figure 1, available online). One or more genes were altered in 25 atients (22.7%). There was no mrna downregulation in T-lymhocyte markers. When the mutual exclusivity and co-occurrence among these four biomarkers was investigated, the HER2 gene showed a tendency towards mutual exclusivity with CD4 (=.227; log odds ratio = -.464), CD8 ( =.29; log odds ratio = -.383), and FOXP3 (=.328; log odds ratio = -.32). However, a tendency towards co-occurrence among T-lymhocyte markers was shown in whole samles (<.1; log odds ratio, > 3). The TCGA Provisional dataset also included clinical HER2- associated variables and among them, variables of IHC-HER2 and HER2 FISH status were relatively comlete data filled u. According to the clinical variables of IHC-HER2 and HER2 FISH status, the mrna exression of T-cell markers was derived (Sulementary Figure 2, available online) and there was no significant association between T-lymhocyte marker exression and clinical HER2 information. After downloading the mrna exression data of T-lymhocyte markers and clinical HER2 information data of IHC-HER2 and HER2 FISH status, the association between T-lymhocyte

4 388 Changro Lee, et al. markers was reinvestigated in HER2-ositive tumors (Figure 1). According to the combined data of IHC-HER2 and HER2 FISH status, 198 out of 1,1 samles (18.%) were clinically categorized as HER2-ositive tumors. The TCGA dataset included 759 (59.%) HER2-negative tumors and 143 (13.%) cases with missing HER2 data. Pearson correlation coefficient between CD4 and CD8, CD4 and FOXP3, and CD8 and FOXP3 was.585 (<.1),.695 (<.1), and.62 (<.1), resec- 1, R 2 linear=.538 1, R 2 linear=.539 CD8A mrna (RNA Seq V2 RSEM) (log) 1, 1 1 FOXP3 mrna (RNA Seq V2 RSEM) (log) , 1, 1 1, 1, CD4 mrna (RNA Seq V2 RSEM) (log) A CD4 mrna (RNA Seq V2 RSEM) (log) B 1, R 2 linear=.512 FOXP3 mrna (RNA Seq V2 RSEM) (log) , 1, CD8A mrna (RNA Seq V2 RSEM) (log) C Figure 1. Association between CD4, CD8, and forkhead P3 (FOXP3) in human eidermal growth factor recetor 2 (HER2)-ositive breast cancers. Among 198 atients with HER2-ositive breast cancer considering immunohistochemistry-her2 and HER2 fluorescence in situ hybridization status variables from the The Cancer Genome Atlas Provisional dataset, the association between (A) CD4 and CD8A, (B) CD4 and FOXP3, and (C) CD8A and FOXP3 is drawn. RSEM=RNA-Seq by Exectation Maximization; V2=version 2. Table 1. Survival analysis according to T-lymhocyte markers using different cutoff levels in HER2-ositive cancer defined by category of HER2 status from the Kalan-Meier lotter Slit atients CD4 CD8 FOXP3 er quartile 42: : : er tertile 55: : : Median 84: : : Uer tertile 113: : : Uer quartile 126: : : HER2= human eidermal growth factor recetor 2; HR=hazard ratio; CI=confidence interval. *Number of atients was resented as atients having low exression versus those having high exression; Hazard ratio of atients having high exression of biomarker.

5 T-lymhocyte Markers in HER2-Positive Breast Cancer 389 tively. The corresonding Searman s rho values were.75 (<.1),.761 (<.1), and.713 (<.1), resectively. Next, the rognostic value of T-lymhocyte markers in HER2-ositive breast cancer was examined using the Kalan- Meier lotter. Since the clear cutoff oints of T-cell markers were not determined, the relase-free survival according to different levels of T-lymhocyte markers was analyzed. Results of the survival analysis using a single marker for 168 HER2-ositive cancers defined by HER2 status are summarized in Table 1 and for 28 HER2-ositive subtyes defined by intrinsic subtye in Table 2. In HER2-ositive cancers defined by HER2 status, as cutoff levels of biomarker increased, the hazard ratio gradually increased in CD4, increased and then decreased in CD8, and gradually decreased in FOXP3. However, there was no statistical significance of T-lymhocyte markers on survival (Table 1). In the HER2-ositive intrinsic Table 2. Survival analysis according to T-lymhocyte markers using different cutoff levels in HER2-ositive subtye defined by category of intrinsic subtye from the Kalan-Meier lotter Slit atients CD4 CD8 FOXP3 er quartile 52: : <.1 38: er tertile 69: <.1 69: : Median 14: : : Uer tertile 139: : : Uer quartile 156: : : HER2= human eidermal growth factor recetor 2; HR=hazard ratio; CI=confidence interval. *Number of atients was resented as atients having low exression versus those having high exression; Hazard ratio of atients having high exression of biomarker HR=.35 (.21.57) log-rank < HR=.38 (.24.61) log-rank < HR=.63 ( ) log-rank = Exression.2 Exression.2 Exression A B C Exression HR=.41 (.23.75) log-rank = D Exression HR=.34 (.17.68) log-rank = E Figure 2. Survival curves using a multigene classifier in human eidermal growth factor recetor 2 (HER2)-ositive subtye defined by category of intrinsic subtye from the Kalan- Meier lotter. Plots were derived from the Kalan- Meier lotter. Intrinsic subtyes are based on the 213 St. Gallen criteria using the exression of HER2, Estrogen recetor (ER), and Ki- 67 in the Kalan-Meier lotter and HER2- ositive intrinsic subtye is defined as HER2+/ ER. In 239 atients with the HER2-ositive intrinsic subtye, atients are slit by (A) lower quartile, (B) lower tertile, (C) median, (D) uer tertile, and (E) uer quartile. HR=hazard ratio.

6 39 Changro Lee, et al. subtye, higher exression of CD4 and CD8 was significantly associated with favorable survival irresective of cutoff levels (Table 2). Only when the atients were slit by uer tertile and uer quartile of CD4, log-rank -value was not statistically significant. Irresective of cutoff levels, the hazard ratio of higher FOXP3 exression was less than.1, resulting in no statistical significance. Since the T-lymhocyte markers were significantly co-occurring in rior TCGA Provisional analysis, we reanalyzed survival using a multigene classifier for considering T-lymhocyte markers all together. Similar to the results in Table 1, there was no statistical significance in HER2-ositive cancers defined by HER2 status (log-rank >.5) (Sulementary Figure 3, available online). In the HER2-ositive intrinsic subtyes, however, higher exression of T-lymhocyte markers was associated with favorable survival irresective of cutoff levels excet for the median value (Figure 2). Finally, we exlored the clinical imlications of T-cell markers on the resistance to HER2-directed theray by using tissue blocks of revious atient cohorts. Of the 47 tissue blocks, 3 (63.8%) were obtained from the breast. Tissue samles were obtained from locoregional sites in nine cases (19.1%). The lungs, brain, and bone or soft tissues were obtained in three (6.4%), three (6.4%), and two cases (4.3%), resectively. Positive IHC staining of the biomarkers are shown in Figure 3. Clinicoathological characteristics and exression of T-lymhocyte markers are resented according to the resonse to targeted theray in Table 3. When the ositive criteria were defined as CD4 3+, CD8 2+, and FOXP3 1% considering frequency of marker exression in this study, all good resonders showed ositive exression of T-lymhocyte markers one or more, although there was no statistical significance. Among oor resonders, 18-matched baseline and resistant tumor samles before and after targeted theray were comared considering combined 3 T-lymhocyte markers status. At baseline, four atients showed negative results for all 3 T- lymhocyte markers exression but changed to ositive status Table 3. Comarison of T-lymhocyte markers and clinicoathological characteristics between atients with good and oor resonse to targeted theray Characteristic Poor resonder grou (n=24) No. (%) Good resonder grou (n=5) No. (%) Total -value* Age (yr) 46.4± ± ± Initial stage II 11 (45.8) 2 (4.3) 13 (44.8) >.999 III 1 (41.7) 3 (6.) 13 (44.8) IV 3 (12.5) 3 (1.3) Hormone recetors ER (+) or PR (+) 13 (54.2) 3 (6.) 16 (55.2) >.999 ER ( )/PR ( ) 11 (45.8) 2 (4.) 13 (44.8) Regimens of targeted theray Trastuzumab based 16 (66.7) 4 (8.) 2 (69.) >.999 TKI±trastuzumab 8 (33.3) 1 (2.) 9 (31.) CD (8.3) 2 (6.9) (41.7) 1 (34.5) (5.) 5 (1) 17 (58.6) CD (41.7) 2 (4.) 12 (41.4) (54.2) 2 (4.) 15 (51.7) 3+ 1 (4.2) 1 (2.) 2 (6.9) FOXP3 Negative (% 9%) 15 (62.5) 3 (6.) 18 (62.1) >.999 Positive ( 1%) 9 (37.5) 2 (4.) 11 (37.9) CD4/CD8/FOXP3 All-negative 5 (2.8) 5 (17.2).553 Either-ositive 19 (79.2) 5 (1) 24 (82.8) ER=estrogen recetor; PR=rogesterone recetor; TKI=tyrosine kinase inhibitors; FOXP3=forkhead P3. *Fisher exact test; Mean±SD; Indeendent two samles t-test. A B C Figure 3. Immunohistochemical exression of CD4, CD8, and forkhead P3 (FOXP3) in human eidermal growth factor recetor 2 (HER2)-ositive breast cancer tissues ( 2). Immunohistochemical staining reveals that tumor infiltrating lymhocytes adjacent to cancer cells (black arrows) are reactive to CD4 (red arrow) (A), CD8 (red arrow) (B), and FOXP3 (red arrow) (C).

7 T-lymhocyte Markers in HER2-Positive Breast Cancer 391 of either biomarker at recurrent or metastatic samle. At baseline, 14 cases were ositive for 1 T-lymhocyte markers and remained ositive in 1 cases at recurrent or metastatic samles. However, four atients with ositive status of either marker at baseline changed to negative exression of all T-cell markers to target recurrent or metastatic samles (Fisher exact test, =.524). There was no case of all negative markers at baseline and recurrent samles both concordantly. Discordant findings of combined markers between baseline and resistant tumors were noted in eight of 18 samles (44.4%). DISCUSSION In the resent study, HER2 was mutually exclusive with T- lymhocyte markers and significant correlation between T- cell markers was observed in HER2-ositive tumors. According to the web-based bioinformatics analysis, there was no statistical imortance of individual or combined T-cell markers on survival in clinically determined HER2-ositive tumor irresective of cutoff levels of biomarkers. In the HER2-ositive intrinsic subtye, however, CD4 and CD8 were determined to have significantly ositive association with survival; statistical significance remained when combined T-lymhocyte markers were considered irresective of cutoff levels. Although small clinical samle size made it difficult to show the statistical imortance of IHC study, good resonders to neoadjuvant chemotheray with targeted theray may be associated with ositive exression of combined T-lymhocyte markers. Discordance of combined markers between the baseline and resistant tumors in aroximately half of the samles suggested that individualized resistance mechanisms might be involved in HER2-directed theray. A recent systematic review showed that the magnitude of TILs was variable within and between breast cancer subtyes and that lymhocyte-redominant breast cancer, CD8+ T- cells, and FOXP3+ TILs were 16% (range, 11% 24%), 61% (range, 4% 83%), and 67% (range, 61% 74%) in HER2-ositive subtye, resectively [21]. In the resent study, mrna exression of CD4, CD8, and FOXP3 was not different according to HER2 status of the TCGA dataset, but there was a significant correlation between T-cell markers. In addition, revious evidence have suorted the rognostic and redictive roles of TILs in breast cancers [13,14,22,23]. Our study also suggested a ossible rognostic role of T-lymhocyte markers measured at mrna levels in HER2-ositive tumors. However, atient selection remains a key issue, and significance was shown only in ER-negative and HER2-ositive subtye defined by intrinsic subtye. There should be an agreement on the standardization of some methodological roblems before the alication of T- cell markers in routine clinical ractice. Multicenter international ring studies by the international Immuno-oncology Biomarker Working Grou demonstrated that reroducible evaluation of TILs using slides is feasible and the softwareguided image evaluation is useful [24]. Recommendations by an international TILs working grou described that it is still unknown whether RNA or rotein classification of TILs shows better erformance of rognostic or redictive value than slide morhology and that further evidence of detecting secific suboulations by IHC is necessary [14]. In addition, our mrna analysis could not discriminate the stromal and intratumoral TILs status although TILs Working Grou recommended the ercentage in the stromal comartment. More evidence is required for the clinical use of TILs. A consensus should be reached on the cutoff levels for defining the clinically beneficial grous of TILs. The international TILs Working Grou 214 recommended no formal thresholds for clinically relevant TILs because a valid methodology was more imortant [14]. In the resent study, both single and combined T-lymhocyte markers were significant rognostic factors irresective of the cutoff values for mrna exression. However, redictive cutoff levels could not be investigated. The FinHER and NCCTG N9831 studies suggested that increased immune function could be a redictor of resonse to trastuzumab [12,25]. Nevertheless, TILs or immunity status should not be used to select the indications for targeted theray at resent [14]. Several mechanisms of resistance to targeted theraies have been suggested including escae from ADCC; alteration in HER2 (known as 95-HER2); exression of other growth factor signal recetors such as eidermal growth factor recetor, HER3, and insulin-like growth factor-1 recetor; cross-talk between ER and HER2 signaling; and uregulation of PI3K- AKT-mTOR athway [6,7,26]. The discordance in the exression rates of T-lymhocyte markers between rimary and metastatic tissues have not been fully understood, but our study demonstrated that half of the samles showed discordant findings. Although it is likely that different comlex mechanisms coexist, most studies evaluated each biomarker including the ones in the resent study; overcoming this hurdle is a future challenge [7]. Limitations of the resent study were that mrna exression and IHC staining were currently not recommended for assessing T-lymhocyte marker exression, and that indeendent validation and large clinical samle size were required for verifying their roles as rognostic and redictive biomarkers. There are many other suboulations of immune cells or various immune markers including CD3; however, we have only

8 392 Changro Lee, et al. alied 3 markers. Further studies are required for the alication of immune function to daily ractice. In conclusion, the resent study attemted to investigate the clinical imlications of CD4, CD8, and FOXP3 on the survival of atients with HER2-ositive breast cancers using different atient selection criteria and various cutoff levels based on a web-based database. A certain roortion of T-lymhocyte marker exression between rimary and resistant tissues resented discordant findings, which suggested different and ersonalized resistance mechanisms to targeted theray. In the era of wide use and focus on develoment of HER2-directed theraies, the association of immune function with HER2 athway and setting u diagnostic and theraeutic strategies for overcoming resistance mechanisms to HER2- targeted theraies is imerative. CONFLICT OF INTEREST The authors declare that they have no cometing interests. REFERENCES 1. 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