PD-L1 Expression and Intratumoral Heterogeneity ... - IngentaConnect

ORIGINAL ARTICLE

PD-L1 Expression and Intratumoral Heterogeneity Across Breast Cancer Subtypes and Stages An Assessment of 245 Primary and 40 Metastatic Tumors Erik A. Dill, MD,* Alejandro A. Gru, MD,* Kristen A. Atkins, MD,* Lisa A. Friedman, BA,w Margaret E. Moore, BS,w Timothy N. Bullock, PhD,* Janet V. Cross, PhD,* Patrick M. Dillon, MD,z and Anne M. Mills, MD*

Abstract: Tumor expression of programmed cell death ligand 1 (PD-L1) is associated with immune evasion in a variety of malignancies, including a subset of triple-negative breast carcinomas, and may mark cancers as susceptible to PD-1/PD-L1 inhibitor therapies. We herein characterize PD-L1 expression in breast cancers across the full range of histomorphologies and investigate its intratumoral heterogeneity and fidelity across primaries and metastases. A total of 245 primary and 40 metastatic (20 nodal, 20 distant) breast carcinomas were evaluated with PD-L1 immunohistochemistry on tissue microarray. Tumor PD-L1 staining was seen in 12% of all primaries including 32% of triple-negative cancers. Staining was common in ductal cancers with medullary (54%), apocrine (27%), and metaplastic features (40%). However, diffuse (> 50%) staining was rare (2% of all cancers and 5% of triple negatives). Immune staining was seen in 29% of all primaries and 61% of triple negatives. Tumor expression of PD-L1 was conserved in 94% of matched primary/ metastasis pairs, while immune staining showed fidelity in 71%; the remaining cases acquired PD-L1 immune cell expression in the metastasis. Only half of cases with positive tumor staining showed concordance across all analyzed cores. These data demonstrate that PD-L1 expression is prevalent among highgrade, hormone receptor–negative breast cancers with a range of histomorphologies and shows fidelity between primary and metastatic sites in treatment-naive cancers, although acquisition of immune PD-L1 staining in metastases is not uncommon. There is considerable intratumoral heterogeneity in PD-L1 expression, undermining the suitability of core biopsy in the determination of PD-L1 status. Clinical trials are needed to determine PD-L1 staining thresholds required for therapeutic response, as diffuse staining is rare.

From the Departments of *Pathology; zOncology; and wSchool of Medicine, University of Virginia, Charlottesville, VA. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Anne M. Mills, MD, Department of Pathology, University of Virginia, Charlottesville, VA 22908 (e-mail: amm7r@ virgnia.edu). Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

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Key Words: breast carcinoma, breast cancer, triple-negative breast cancer, PD-L1, PD-L1 inhibitors, checkpoint inhibitors, immunotherapy (Am J Surg Pathol 2017;41:334–342)

BACKGROUND The role of the immune system in facilitating tumor clearance is evident in a host of malignancies, including breast cancer. Conversely, dysfunctional immune responses are known to permit tumor progression: while the presence of a robust cytotoxic T-cell response is associated with good prognosis in breast carcinoma, inhibition of this response allows unchecked tumor growth.1–6 One mechanism of immune suppression that has been implicated in tumor progression is the programed cell death-1 (PD-1) pathway.7–9 The cell surface protein PD-1 is normally expressed on a variety of immune cells and interacts with its ligands (PD-L1 and PDL2) to tamp down cytotoxic T-cell activity and stimulate regulatory T-cell development, thereby extinguishing the immune response.10 A normally functioning PD-1-PD-L1 pathway plays an important role in quenching immune reactions following infection and other inflammatory stimuli, providing protection against immunopathology. When this pathway is co-opted by malignancy, however, it can prevent host control of tumor growth and enable tumor progression.7 One mechanism by which malignant cells capitalize on the PD-1-PD-L1 immune inhibitory pathway is through tumor cell expression of PD-L1.7,9 Tumoral PD-L1 expression is of considerable clinical interest due to the recent development of PD-1/PD-L1 blocking antibodies, which show robust efficacy and durable clinical response in a variety of malignancies including melanoma, bladder carcinoma, and lung carcinoma.7,11–14 PD-L1 expression has recently been demonstrated in a subset of breast carcinomas, with increased incidence among tumors with poor prognostic variables such as large size, high grade, and hormone receptor negativity.4,15–19 There is also interest in PD-L1 expression by peritumoral and tumor-infiltrating Am J Surg Pathol

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lymphocytes and macrophages, which may play a role in tumoral immune evasion and response to anti-PD-L1 therapies. Although PD-L1 expression has been clearly identified in a subset of triple negative and Her2-amplified carcinomas as well as occasional hormone receptor– positive ductal and lobular cases, it has not been well studied in the full spectrum of morphologic subtypes.4,15,18 Furthermore, PD-L1 variability across tissue cores collected from different regions of the same tumor has not been thoroughly investigated in breast cancer, nor have PD-L1 expression patterns been well studied in matched primary and metastatic breast tumors. Assessing staining in paired metastases and primaries will address whether PD-L1 acquisition plays a role in the permission of metastasis in immunogenic cancers. Because documentation of PD-L1 expression is required for admission into PD-L1 inhibitor trials in a variety of organs, clarifying the distribution of staining within breast primaries and metastases is of clinical importance as these therapies appear on the horizon for breast cancer. We herein characterize PD-L1 immunohistochemical expression across 245 cases of primary breast carcinoma and 40 cases of metastatic breast carcinoma representing a variety of subtypes, grades, and stages using formalinfixed, paraffin-embedded tissues on tissue microarrays (TMAs).

MATERIALS AND METHODS This study was approved by the institutional review board of The University of Virginia.

Case Selection and TMA Construction Cases were evaluated on 4 previously constructed TMAs containing archival formalin-fixed, paraffinembedded tissues from 245 invasive primary breast carcinomas, 20 nodal metastases, and 20 distant metastases. Control tissues from kidney, liver, and placenta were also present. The arrays contained 4 replicate 0.6 mm cores from each case with samplings from different areas within the original tumor section including the tumor periphery and center. In some instances, one or more of the cores was either lost entirely or failed to contain tumor on the material available for staining; the number of available tumor-containing cores was, therefore, recorded for each case. The arrays were enriched to include the spectrum of tumor stages and histologic subtypes. All primary tumors and all nodal metastases represented on the TMAs were treatment naive. Seventeen of 20 distant metastases occurred after primary resection and therapy (mean time since primary diagnosis: 4.5 y), whereas 3 were identified at the time of primary diagnosis and were treatment naive. Sixteen of the nodal metastases and 1 lung metastasis were matched to concurrent primary tumors also present on the arrays: all matched cases were treatment naive in both locales. Clinicopathologic information including tumor histologic type, grade, stage, and hormone receptor status Copyright

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was obtained on all cases; ER/PR+ cases were classified as Luminal A-like, ER/PR/HER2+ cases were classified as Luminal B-like, hormone receptor–negative cases with HER2 amplification were classified as HER2+, and hormone receptor/HER2 cases were classified as triple negative. Germline BRCA mutation status was obtained whenever possible.

Immunohistochemistry Immunohistochemical staining for PD-L1/CD274 (SP142, rabbit IgG, dilution 1:200, catalog #M4420; Spring Bioscience, Pleasanton, CA) was performed on all TMAs. PD-L1 immunohistochemical staining was scored in both the tumor and the peritumoral immune compartment (eg, the “immune stroma”). Tumor staining was classified positive when clear membranous staining was present in Z1% of tumor cells. Staining extent was further characterized in the following subcategories: 1% to 5%, 6% to 10%, 11% to 25%, 26% to 50%, and >50%. The 1% threshold for positivity was selected based on data demonstrating clinical response to PD-L1 inhibition at this expression level in some cancers, and percentage categories were further selected to include the cutoffs for all available PD-L1 inhibitors.12,20 Immune microenvironment (eg, “immune stromal”) staining was scored as positive when >5% of peritumoral and intratumoral immune cells (including lymphocytes and macrophages) showed PD-L1 reactivity and was further subdivided by extent as 5% to 10%, 11% to 25%, 26% to 50%, and >50%. The 5% lower limit was selected because single, scattered PD-L1-positive inflammatory cells were observed in benign control tissues. Furthermore, because scattered background PD-L1 staining can be seen in normal nodal tissue, assessment of inflammatory PD-L1 positivity within nodal metastases was limited to cells intimately admixed with the metastatic tumor (as opposed to both tumor infiltrating and peritumoral cells, as was the case with assessment in primary and distant metastatic assessments). The number of positive cores was recorded for each case to facilitate assessment of staining heterogeneity.

RESULTS PD-L1 Expression in Primary Tumors Tumor cell expression of PD-L1 was observed in 12.2% (30/245) of primary breast cancers. Diffuse (> 50%) staining was identified in 20% (6/30) of positive cases; 23.3% (7/30) showed 25% to 50% staining, 20% (6/30) showed 10% to 25% staining, and 36.7%, (11/30) showed 5% to 10% staining. The 30 PD-L1-positive cases consisted predominantly of ductal cancers, including both conventional ductal cases and those with medullary, apocrine, neuroendocrine, and metaplastic features. Expression in lobular cancers was rare (Fig. 1 and Table 1A). Expression was most common in high-grade tumors with 30.5% of grade 3, 2.7% of grade 2, and 3.9% of grade 1 cancers showing tumoral staining (Table 1B). PD-L1 positivity was also most frequently observed in

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FIGURE 1. Tumoral PD-L1 expression in a variety of histology subtypes. Tumor cells were scored as positive when circumferential staining was present in at least 1% of cells. A and B, Triple-negative ductal carcinoma (not otherwise specified) demonstrating strong diffuse membranous staining. This diffuse staining pattern was relatively rare in the series, accounting for 6 of 30 positive cases. C and D, Ductal carcinoma with apocrine features showing membranous staining in a subset of tumor cells. E and F, Metaplastic carcinoma demonstrating membranous staining in a subset of tumor cells. Of note, this tumor expressed PD-L1 primarily in the less spindled areas (as depicted here); the frankly spindled regions showed only rare positive cells. G and H, Lobular carcinoma with focal membranous PD-L1 positivity. This was among the only lobular cancers to express PD-L1.

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TABLE 1. PD-L1 Expression in Breast Carcinomas n/N (%) Tumor PD-L1

Immune Stromal PD-L1

(A) PD-L1 expression in primary tumors by morphologic subtype Morphologic subtype Ductal, conventional 13/157 (8.2) 44/157 (28) Ductal with medullary 6/11 (54.5) 11/11 (100) features Ductal with apocrine 3/11 (27.2) 4/11 (36.4) features Lobular 2/23 (8.7) 3/23 (13) Pleomorphic lobular 2/14 (14.3) 2/14 (14.3) Neuroendocrine 2/2 (100) 2/2 (100) Micropapillary 0/2 (0) 0/2 (0) Metaplastic 2/5 (40) 4/5 (80) Mucinous 0/20 (0) 1/20 (5) (B) PD-L1 expression in primary tumors by tumor grade Grade 1 2/51 (3.9) 4/51 (7.8) 2 3/112 (2.7) 16/112 (14.3) 3 25/82 (30.5) 51/82 (62.1) (C) PD-L1 expression by ER/PR/HER2 status* ER/PR/HER2 status Luminal A-like 8/161 (5) 26/161 (16.1) Luminal B-like 3/20 (15) 7/20 (35) 2/4 (50) 2/4 (50) HER2+ Triple negative 18/57 (31.6) 35/57 (61.4) (D) PD-L1 expression by stagew Stage T1 18/143 (12.6) 36/143 (25.2) T2 11/78 (14.1) 29/78 (37.1) T3 0/15 (0) 2/15 (13.3) T4 1/5 (20) 1/5 (20) N+ 2/20 (10) 10/20 (50) M+ 2/20 (10) 10/20 (50) (E) Expression variability across cores for PD-L1-positive primary and metastatic tumors Positive cores 100% (4/4, 3/3, 2/2, 1/1) 17/34 (50) 54/91 (59.3) > 66% (2/3, 3/4) 9/34 (26.5) 10/91 (11) 50% (2/4, 1/2) 6/34 (17.6) 17/91 (18.7) < 34% (1/3, 1/4) 2/34 (5.9) 10/91 (11) (F) Germline BRCA status BRCA mutated 3/6 (50) 6/6 (100) BRCA wildtype 7/16 (43) 13/16 (81) *Analysis excludes 3 primaries for which hormone receptor/HER2 status was unknown. wAnalysis excludes 4 primaries for which tumor stage was unknown.

triple-negative cancers and HER2+ cancers; however, occasional luminal A-like cancers also stained (Table 1C). Of note, 3 of 6 cancers with diffuse (> 50%) PD-L1 staining were high-grade, triple-negative cancers while the other 3 were high-grade cancers showing ER/PR+ (luminal A-like, 2 cases) and ER/PR/HER2+ (luminal B-like, 1 case). Tumoral PD-L1 expression was observed in 12.6% of T1, 14.1% of T2, and 20% of T4 tumors; no T3 tumors showed tumor staining (Table 1D).

PD-L1 Expression in Primary Tumor-associated Inflammatory Cells Tumor-associated inflammatory cell (eg, immune stromal) expression of PD-L1 was observed in 29% (71/ Copyright

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245) of primary breast cancers. Most PD-L1-positive cases showed 1% to 10% staining (40.8%, 29/71), whereas 31% (22/71) showed 10% to 25% staining, 19.7% (14/71) showed 25% to 50%, and 8.5% (6/71) showed >50% immune stromal staining. The 71 positive cases spanned a range of histologies paralleling those observed in the tumoral staining group (Fig. 2 and Table 1A). PD-L1 immune stromal positivity was most common in high-grade tumors, 63% of which showed staining (Table 1B). The majority (61.4%) of triple-negative cancers showed PD-L1 positivity within tumor-associated inflammation (Table 1C). Immune stromal staining for PD-L1 was observed across the spectrum of tumor stages with positivity in 25.2% of T1, 37.1% of T2, 13.3% of T3, and 20% of T4 tumors (Table 1D).

PD-L1 Expression in Metastases Tumor cell expression of PD-L1 was observed in 10% (2/20) of nodal and 10% (2/20) of distant metastases. Of the matched treatment-naive primary and metastatic tumors, 94% (16/17) of cases showed concordant staining between the primary and metastatic tumor (Fig. 3). One case demonstrated a loss of PD-L1 positivity from the primary to the metastatic tumor; however, the primary tumor showed only focal staining, therefore, sampling limitations may play a role. In total, 70.6% (12/17) of the matched cases showed concordant staining between the primary and metastatic tumor-associated inflammatory cells. Five cases (4 nodal metastases and 1 lung metastasis) demonstrated a gain of PD-L1 immune stromal positivity in the site of metastasis.

PD-L1 Expression Across Replicate Cores PD-L1 positivity varied across replicate cores from the same tumors with 50% (17/34) of cases with tumoral PD-L1 staining (including primaries and metastases) demonstrating 100% concordance between all analyzed cores, 26.5% (9/34) showing 66% to 75% concordance (positivity in 2/3 or 3/4 cores), 17.6% (6/34) cases showed 50% concordance, and 5.9% (2/34) of cases showing 25% to 34% concordance (positivity in 1/3 or 1/4 cores) Table 1E. Discrepant results across cores were most common in cases showing lower levels (< 25% staining extent) of PD-L1 positivity, whereas 71.4% (5/7) of cases with 25% to 50% staining and 83.3% (5/6) of cases with diffuse (> 50%) tumoral staining were consistent across all sampled cores. Of the 91 primary and metastatic cases showing PDL1 staining in the immune stroma, PD-L1 expression positivity between cores is as follows: 59.3% (54/91) of cases demonstrated 100% concordance between all analyzed cores, 11% (10/91) of cases showed 66% to 75% concordance, 18.7% (17/91) cases showed 50% concordance, and 11% (10/91) of cases showed 25% to 34% concordance Table 1E.



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FIGURE 2. PD-L1 expression in immune stroma. Staining in immune stroma was scored as positive when >5% of tumorassociated inflammatory constituents (including lymphocytes and macrophages) were positive. These 2 high-grade ductal carcinomas (A-B and C-D) demonstrate strong PD-L1 immunostaining with lymphocytes and macrophages adjacent to tumor cell nests, but show only focal, 50% PD-L1 staining for eligibility, but a similar study of the drug nivolumab failed to show benefit in a cohort in which 5% staining was used as the cutoff for inclusion.25,26 The clinically relevant threshold for PD-L1 positivity has not been established for breast



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cancer, but it is reasonable to encourage caution regarding the promise of PD-L1 inhibitors for breast cancer patients as extrapolation from experience in the lung indicates that only a subset may benefit. This caution should be amplified by the fact that, while less toxic and better tolerated than many conventional chemotherapeutics, PD-L1 inhibitors have been associated with a host of immune dysregulatory complications including colitis, myocarditis, and inflammatory dermatoses.13,27,28 Tumoral PD-L1 expression generally showed fidelity across the matched primary and metastatic samples from treatment-naive patients, with only a single case showing discrepant results. There is therefore no evidence in this series to suggest a primary role for tumoral PD-L1 acquisition in promoting metastatic spread in the absence of neoadjuvant therapy. This is concordant with the work by Cimino-Mathews et al,23 who identified tumor staining discordance in only 1 of 26 matched primary and metastatic breast cancer. Immune stromal expression was more variable across matched primary and metastatic sites, with 29% of cases gaining PD-L1-positive peritumoral immune cells in the site of metastasis. Of note, one of the cases that acquired PD-L1-positive immune stromal cells in the site of metastasis was the only matched lung metastasis in this series; this was also observed in both the lung metastases in the study by Cimino-Mathews et al.23 Given that a significant subset of cases apparently acquires PD-L1positive immune infiltrates at metastatic sites, it may be that the recruitment of such an infiltrate facilitates the spread of breast carcinoma through immune escape. This thesis is supported in part by the finding that PD-L1positive immune stromal cells seem to play a role in the progression of ductal carcinoma in situ.29 However, the significance of PD-L1 expression in peritumoral immune stromal cells remains poorly understood and does not presently factor into patient selection for anti-PD-L1 therapy. These results may have implications about the need for repeat testing in metastases from treatment-naive patients whose primary tumors have previously been evaluated for PD-L1 expression. If clinical work demonstrates that only tumoral PD-L1 staining identifies cancer patients as candidates for PD-L1 inhibition, our data indicate that repeat testing of treatment-naive metastases may not be warranted. However, repeat testing may be indicated if immune cell PD-L1 positivity is proven to predict PD-L1 inhibitor response. It is critical that clinical trials involving PD-L1 inhibitors enlist careful assessment of PD-L1 staining patterns and thresholds within both the tumor and the immune stroma. Further work is needed to assess PD-L1 status in pretreatment and posttreatment specimens to ascertain whether treatment induces PD-L1 expression in surviving/recurrent tumor clones. This study represents the first systematic assessment of PD-L1 expression heterogeneity in breast cancer. By assessing expression across multiple replicate cores from the same tumor, we were able to determine how often sampling localization influenced the expression results. Only 50% of cases with positive tumor cell staining

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demonstrated positivity across all the sampled cores, while nearly a quarter were positive in half or fewer of the sampled cores. This sampling heterogeneity was expectedly centered on tumors that had lower overall levels of staining; cases with higher levels of staining showed more uniform reactivity across cores, with 83.3% (5/6) of cases with >50% staining showing complete concordance. Immune compartment staining was similarly variable although positive cases with complete concordance across cores were slightly more common (59.3%). This finding suggests that PD-L1 staining has a relatively low negative predictive value when only a single region of tumor is sampled; therefore, caution should be exercised when interpreting this stain on small biopsy specimen if lower levels of PD-L1 expression prove to be clinically actionable. In contrast, core biopsy results may typically be sufficient if clinical trials suggest that a benefit to PD-L1 inhibition is observed only for tumors with diffuse staining. Further work is needed to assess the geographic distribution of PD-L1 expression in breast cancer as so-called “marginal” tumor staining patterns—that is, staining at the periphery of tumor nests—have been shown to be clinically significant in cervical cancers.30 This is also true for peritumoral immune cell positivity localized to the invasive front of the tumor, a pattern that has been observed in upper gastrointestinal adenocarcinomas.31 Detailed analysis of PD-L1 expression patterns in whole tissue sections of breast carcinoma will therefore be of interest. A limitation of this study is that PD-L1 expression was based on a single (albeit well-characterized and widely validated) antibody clone (SP142; Spring Bioscience). The current status of PD-L1 immunohistochemistry is fraught with reports of inconsistent results across different antibodies and the Food and Drug Administration mandate that specific clones/companion diagnostic kits be used to determine eligibility for individual drugs (such as Dako’s 22C3 pharmDX PD-L1 antibody and the drug pembrolizumab).32,33 Given that there is presently no current Food and Drug Administration requirement for a specific PD-L1 clone in breast cancer, we selected the SP142 antibody for the current study because this antibody has been used in other studies on PD-L1 in breast cancer15 and has been used with good success in our clinical laboratory. Another limitation is the absence of direct correlation with germline BRCA mutation status for many of the patients. BRCA1/2 mutations have been associated with increased tumor immunogenicity and regulatory T-cell modulated immune escape in breast cancers, and have been affiliated with higher PD-L1 expression in serous ovarian cancers.16,34 BRCA1/2-mutated cancers are known to demonstrate high neoantigen loads and this may represent a situation analogous to mismatch repair deficient colorectal and endometrial tumors, which have been shown to express PD-L1 as a presumed counterbalance to the high levels of antitumoral cytotoxic T cells they recruit through this continuous production of novel tumoral antigens.16,35–37 In this series, a slight trend Copyright

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toward increased PD-L1 positivity was observed in patients with known BRCA mutations; however, this was not statistically significant. Further work specifically addressing PD-L1 expression in a larger cohort of known BRCA1/2-mutated cancers will be of interest to determine whether these tumors have a unique vulnerability to inhibitor therapies against PD-L1. In summary, we herein demonstrate that some degree of tumoral and immune stromal PD-L1 expression is identified in a subset of breast cancers of a variety of histologic subtypes, but is most common among high-grade, hormone receptor–negative ductal cancers including those with medullary, apocrine, and metaplastic features. However, even among triple-negative cancers, diffuse tumor cell expression is relatively rare (B5%). Tumoral PD-L1 status appears conserved across primary and metastatic sites in treatment-naive cancers; however, the acquisition of a PDL1-positive inflammatory component is not uncommon in metastases. PD-L1 expression by both tumor cells and immune infiltrates is geographically variable across cores from different sites within the same tumor, suggesting that individual biopsies may not be representative of overall PD-L1 status. Further work correlating tumoral and immune stromal PD-L1 expression levels with clinical response to PD-L1 inhibition will be critical, particularly among high-grade, hormone receptor–negative tumors. If response is seen in the setting of any tumor staining and/or any peritumoral immune staining, roughly a third of all breast cancer could be expected to respond to these immunotherapies. However, if diffuse tumor cell staining is required for clinical benefit, a much smaller proportion of breast cancer patients will be rational candidates.


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ACKNOWLEDGMENT The authors would like to acknowledge the skill and expertise of the University of Virginia Biorepository and Tissue Research Facility in the construction of Tumor Microarrays. REFERENCES 1. Loi S, Michiels S, Salgado R, et al. Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: Results from the FinHER trial. Ann Oncol. 2014;25:1544–1550. 2. Denkert C, Loibl S, Noske A, et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol. 2010;28:105–113. 3. Rody A, Holtrich U, Pusztai L, et al. T-cell metagene predicts a favorable prognosis in estrogen receptor-negative and HER2positive breast cancers. Breast Cancer Res. 2009;11:R15. doi: 10.1186/bcr2234. 4. Sabatier R, Finetti P, Mamessier E, et al. Kinome expression profiling and prognosis of basal breast cancers. Mol Cancer. 2011;10:86-4598-10-86. doi: 10.1186/1476-4598-10-86. 5. Teschendorff AE, Miremadi A, Pinder SE, et al. An immune response gene expression module identifies a good prognosis subtype in estrogen receptor negative breast cancer. Genome Biol. 2007;8:R157. doi: gb-2007-8-8-r157. 6. Ali HR, Provenzano E, Dawson SJ, et al. Association between CD8+ T-cell infiltration and breast cancer survival in 12,439 patients. Ann Oncol. 2014;25:1536–1543. 7. Iwai Y, Ishida M, Tanaka Y, et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immuno-

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