Human Solid Tumors Contain High Endothelial Venules: Association with T- and B-Lymphocyte Infiltration and Favorable Prognosis in Breast Cancer

Published OnlineFirst August 16, 2011; DOI: 10.1158/0008-5472.CAN-11-0431

Cancer Microenvironment and Immunology Research

Ludovic Martinet1,2, Ignacio Garrido1,2,4, Thomas Filleron4, Sophie Le Guellec4, Elisabeth Bellard1,2, Jean-Jacques Fournie3,5, Philippe Rochaix4, and Jean-Philippe Girard1,2

Abstract The mechanisms governing infiltration of lymphocytes into tumors remain poorly characterized, in spite of the critical impact of these cells on patient prognosis and therapeutic responses. High endothelial venules (HEV) are blood vessels found in lymphoid tissues, specialized in lymphocyte recruitment, but their implications in þ human cancer are unknown. In this article, we report the presence of MECA 79 blood vessels displaying all the phenotypic characteristics of HEVs in most of the 319 human primary solid tumors, including melanomas, breast, ovarian, colon, and lung carcinomas, analyzed. Tumor HEVs were specifically located within lymphocyte- þ þ rich areas, and their density within the tumor stroma was a strong predictor of infiltration by CD3 and CD8 T cells as well as B cells. Large-scale flow cytometric and quantitative reverse transcriptase-PCR analyses in freshly operated breast tumors revealed that high densities of tumor HEVs correlated with increased naive, central memory and activated effector memory T-cell infiltration and upregulation of genes related to T-helper 1 adaptive immunity and T-cell cytotoxicity. Finally, in a retrospective cohort of 146 invasive breast cancer patients, we found that high densities of tumor HEVs independently conferred a lower risk of relapse and significantly correlated with longer metastasis-free, disease-free, and overall survival rates. Together, our findings suggest that tumor HEVs function as major gateways for lymphocyte infiltration into human tumors, and may represent attractive targets for cancer diagnosis and therapy. Cancer Res; 71(17); 1–10. 2011 AACR.

Introduction overexpression of the endothelin B receptor on tumor blood vessels functions as a barrier that inhibits lymphocyte adhe- The immune system plays a critical role in tumor surveil- sion to endothelium and restricts lymphocyte infiltration into lance (1), patient's clinical outcome (2), and therapeutic tumors (13). In addition, tumor angiogenesis has been shown response (3, 4). The presence of high numbers of tumor- to induce a downregulation of endothelial cell-adhesion mole- infiltrating lymphocytes, particularly T cells, has been found cules such as intercellular adhesion molecule 1 (ICAM-1; ref. to be a major predictor of favorable clinical outcome in several 14). In contrast, the characteristics of tumor blood vessels solid cancers, including colorectal (5–7), lung (8), and ovarian which facilitate large-scale influx of lymphocytes within carcinomas (9–11). However, the blood vessels and mechan- human tumors are presently unknown. isms governing the recruitment of lymphocytes into tumors High endothelial venules (HEV) are specialized post– remain poorly understood. capillary venules found in lymphoid tissues that support The first critical step in lymphocyte migration from circula- high levels of lymphocyte extravasation from the blood (15, tion into tissue is the adhesion of lymphocytes to vascular 16). HEVs are composed of plump, cuboidal endothelial cells, endothelium (12). Evidence has recently been provided that expressing 6-sulfosialyl Lewis X ligands for the lymphocyte- homing receptor l -selectin (CD62L), that mediate the initial capture and rolling interactions of lymphocytes along the Authors' Affiliations: 1CNRS, IPBS (Institut de Pharmacologie et de vessel wall (15–17). Although HEVs are generally restricted 2 3 Biologie Structurale); UPS, IPBS, and UPS, CPTP, UniversitedeTou- to lymphoid organs, evidence accumulated over the past 25 louse; 4Institut Claudius Regaud; and 5INSERM, U563, Centre de Physio- pathologie de Toulouse-Purpan, Toulouse, France years indicates that blood vessels with HEV characteristics Note: Supplementary data for this article are available at Cancer Research develop in nonlymphoid tissues in many chronic inflamma- Online (http://cancerres.aacrjournals.org/). tory diseases, including rheumatoid arthritis, inflammatory L. Martinet and I. Garrido contributed equally to this study. bowel diseases, chronic gastritis, and autoimmune thyroi- – Corresponding Author: Jean-Philippe Girard, IPBS-CNRS-Universitede ditis (15, 18 20). Toulouse, 205 route de Narbonne, 31077 Toulouse, France. Phone: 33-5- In the present study, we investigated the presence of HEVs 61-17-59-18; Fax: 33-5-61-17-59-94; E-mail: [email protected] within various human primary solid tumors and their associa- doi: 10.1158/0008-5472.CAN-11-0431 tion with lymphocyte infiltration, immune orientation, and 2011 American Association for Cancer Research. clinical outcome in breast cancer.

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þ Materials and Methods date automatic cell count. The density of CD34 blood vessels was calculated by optical counting of vessel numbers on 10 Patients representative tumor fields (0.5 mm2; original magnification This study was approved by the Scientific Review Board of 10). Quantification of vessels and scoring were done by 3 the Institute Claudius Regaud (ICR; Toulouse, France). The independent observers who were blinded to the clinical out- prospective study on HEVs in human solid tumors was con- come. We used the following cutoff points (highest tercile vs. 2 ducted on paraffin-embedded tumor blocks with representa- lowest terciles) to discriminate high and low densities of the þ þ tive tumor areas of 18 primary melanomas, 5 primary colon different cell populations: CD3 T cells, 270 cells/mm2; CD8 þ carcinomas, 5 primary lung carcinomas, 18 primary ovarian T cells, 150 cells/mm2; and CD20 B cells, 135 cells/mm2. carcinomas, and 127 primary breast carcinomas, operated on between 2003 and 2010. The retrospective study was conducted Large-scale flow cytometric analysis with a cohort of 146 unselected, primary, nonmetastatic, Freshly resected breast carcinoma samples were reduced to invasive breast cancer patients operated at the ICR between small fragments and incubated for 30 minutes at 37 Cin 1997 and 1998. Patient characteristics are described in Supple- sterile RPMI-1640 containing Collagenase IV (1 mg/mL; mentary Table S1. None of the patients analyzed in our study Sigma-Aldrich). Total cells were then extracted by mechanical received chemotherapy or radiotherapy before surgery. They dispersion and incubated for 30 minutes at 4 C with anti- did not have previous history of cancer and did not present bodies directed against markers for different immune cells or distant metastasis at the time of surgery. Postsurgical surveil- their isotype-matched controls (Supplementary Table S3). lance of patients was carried out at the ICR according to Analyses were carried out on a 6-color fluorescence-activated general standard practice for breast cancer patients. The cell sorter (LSRII; Beckton Dickinson) with Diva Software median follow-up was 122 months, during which there were (Beckton Dickinson). Overall, 120 combinations of surface 61 relapses including 52 metastatic relapses and 50 deaths. and intracellular markers were used to identify the different populations of tumor-infiltrating immune cells (Supplemen- tary Fig. S1). Heat-map representation of populations of Immunohistochemistry and immunofluorescence immune cells expressed as a percentage of total cells extracted staining from tumor tissues was realized with the use of dChip Soft- Immunohistochemistry was carried out on 5-mm-thick ware (Harvard School of Public Health, Boston, MA). consecutive sections from paraffin-embedded tumor blocks using a Techmate Horizon slide processor (Dako). Details of Quantitative reverse transcriptase-PCR the antibodies, fixatives, and antigen-retrieval methods used An RNeasy Isolation kit (QIAGEN) was used to isolate total are provided in Supplementary Table S2. Briefly, slides were RNA from 20 cryopreserved breast tumor samples with a low incubated with primary antibodies for 1 hour at room tem- or high density of tumor HEVs. The integrity and the quantity perature. Antigen–antibody complexes were visualized using a of the RNA were evaluated using 2100 Bioanalyzer (Agilent peroxidase-conjugated polymer backbone coupled to a sec- Technologies). cDNA was prepared by reverse transcription ondary antibody system (EnVision; Dako) and 3,30-diamino- using superscript VILO cDNA Synthesis Kit (Invitrogen). benzidine chromogen (Dako). For immunofluorescence Reverse transcriptase (RT)-PCR experiments were conducted detection, slides were incubated with secondary antibodies using Power SYBR Green Master Mix with an ABI PRISM coupled to AF-488 or Cy3, diluted in PBS/bovine serum 7300HT (Applied Biosystems) according to the manufacturer's albumin (BSA) 1%, for 1 hour at room temperature and 0 instructions. All reactions were done in duplicate and normal- counterstained with 4 ,6-diamidino-2-henylindole (DAPI). ized to the expression of glyceraldehyde phosphate dehydro- genase (GAPDH). For each gene, relative change in expression Method for cell quantification was calculated by the DDcycling threshold (CT) method as (DCTsampleDCTcontrol Tumor slides stained with MECA-79, anti-CD3, anti-CD20, 2 ) with DCTcontrol ¼ average DCT from and anti-CD8 antibodies were scanned with a high-resolution HEVlow tumors. scanner (NDP Slide scanner; Hamamatsu). Absolute numbers þ of MECA-79 vessels present within the tumor area (mm2) Statistical analysis were quantified for each tumor slide, and the densities of We analyzed 3 main endpoints: metastasis-free survival tumor HEVs (HEV/mm2) were calculated. Because no cutoff rate, which was defined as time from surgery to distant point has been previously described for density of tumor metastases (all other events were ignored for this endpoint); HEVs, we discriminated patients with a high and a low density disease-free survival rate, that is, time from surgery to any of tumor HEVs with the following cutoff points (highest tercile recurrence (local or regional), second breast primary, distant vs. 2 lowest terciles: 0.19 HEV/mm2). Automatic cell counts of metastasis, or death from any cause; and overall survival rate, þ þ þ CD3 , CD20 , and CD8 cells were determined on 10 repre- which was defined as time from surgery to death from any sentative tumor fields (4.4 mm2; original magnification 5; cause. Categorical variables were reported by frequencies and 2,560 2,048 resolution) with Image J Software (NIH, percentages; continuous variables were presented by median þ þ þ Bethesda, MD). Furthermore, CD3 , CD20 , and CD8 cells and range. Comparative analyses between groups were done were counted semiquantitatively (score: 0, 1, 2, and 3 for none, using the Mann–Whitney rank-sum test for continuous low, intermediate, and high density of positive cells) to vali- variables and the c2 test or Fisher's exact test for categorical

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variables. Correlations between continuous variables were epitope, see Supplementary Fig. S2), an HEV-specific mono- evaluated using Spearman's rank correlation test. The clonal antibody which recognizes sulfated ligands for lympho- Kaplan–Meier product-limit estimator was used to display cytes and inhibits lymphocyte–HEV interactions in vivo (17, 21, time-to-event curves for the 3 endpoints. The significance of 22). Vessels expressing high levels of the MECA-79 epitope were various clinical characteristics and density of HEVs was observed in the majority of tumors analyzed (11 of 18 mela- assessed by univariate analysis with the use of the log-rank nomas, 94 of 127 breast, 11 of 18 ovary, 4 of 5 lung, and 4 of 5 þ test. The Cox regression model was applied to determine colon carcinomas; Fig. 1A). In contrast, MECA-79 vessels were whether a factor was an independent predictor of survival not detected in normal control tissues distant from the tumor in multivariate analysis. Two-sided P values of less than 0.05 site. Immunofluorescence staining of tumor sections revealed þ were considered statistically significant. Statistical analyses that MECA-79 vessels are found in tumor areas infiltrated by þ þ þ were done using the STATA 11.0 (STATA Corp) software. both CD3 T cells and CD20 B cells (Fig. 1B). CD3 T cells were frequently seen extravasating or attached to the luminal þ Results surface of the plump, cuboidal MECA-79 endothelial cells, suggesting an active role of these vessels in lymphocyte recruit- Human solid tumors contain blood vessels with HEV ment (Fig. 1C). These endothelial cells expressed HEV markers characteristics HECA-452 (23) and DARC (24), the endothelial cell To analyze the presence of HEVs within human solid adhesion molecule ICAM-1, and pan-vascular markers CD31 tumors, we carried out immunohistochemistry on 173 primary and von Willebrand factor (vWB; Fig. 1D). Interestingly, þ tumor sections with MECA-79 (for details on the MECA-79 tumor-associated MECA-79 vessels were also labeled by

A Breast Melanoma Ovarian Colon Lung

Figure 1. Phenotypic characterization of tumor HEVs. A, blood vessels which express the HEV-specific marker MECA-79 are present in different types of human solid tumors. Immunohistochemical analysis B D with MECA-79 was carried out on DAPI CD20 CD3 IPAD MECA-79 CD31 Merge human melanoma and breast, ovarian, colon, and lung carcinomas. B, MECA-79þ HEVs are present in tumor areas infiltrated by both CD3þ T cells and CD20þ B cells. Consecutive breast tumor sections were DAPI MECA-79 ICAM-I Merge stained by immunofluorescence DAPI CD31 MECA-79 with anti-CD3, anti-CD20, anti- CD31, and MECA-79 antibodies. Counterstaining was done with DAPI. C, CD3þ T cells (red) are seen attached to the luminal surface of MECA-79þ HEVs DAPI HECA-452 DARC Merge endothelial cells (green) and extravasating through the vessel C DAPI CD3 MECA-79 wall (white arrows). D, tumor HEVs express pan-endothelial cell markers CD31 and vWB, endothelial cell-adhesion DAPI MECA-79 G72 Merge molecule ICAM-1, post–capillary venule–specific marker DARC, and HEV-specific markers MECA- 79, HECA-452, G72, and G152. Immunofluorescence staining of breast tumor sections was done with the indicated antibodies. DAPI VWB G152 Merge Counterstaining was done with DAPI. Original magnification (A, 40;B,10; and C and D, 100).

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Figure 2. The density of tumor HEVs predicts T- and B-lymphocyte infiltration in breast cancer. A, representative photograph from digitized tumor slide stained with B C MECA-79 antibody showing HEVs ) 2 ) (arrows) within breast tumor stroma. 2 1,000 10 200 B, absolute number and density of HEVhighhigh N.S. þ (mm

(mm HEV MECA-79 HEVs in the tumor area mber m 150 a 100 1 a for each patient of the retrospective 100 breast cancer cohort (n ¼ 146). The 50 red bar indicates the cutoff value 10 0.1 used to discriminate high and low low HEVlow 0 densities of HEVs (highest tercile vs. 0.01 2 lowest terciles). C, the density of 1 low high þ

HEV/tumor area HEV HEV CD34/tumor are CD34 blood vessels is similar in HEV absolute nu HEVlow and HEVhigh breast tumors. 0 0 The line in the center of each box represents the median value of the low high D HEV HEV distribution, and the upper and lower ends of the box are the upper and MECA-79 CD34 lower quartiles, respectively. D, E tumors containing a high density of tumor HEVs exhibit high levels of P < 0.001 þ þ 3,000 infiltrating CD3 T cells, CD8 2 T cells, and CD20þ B cells. 2,000 Consecutive breast tumor sections from representative HEVhigh and 1,000 HEVlow tumors were analyzed by CD3 CD3/mm 0 immunohistochemistry with MECA-79, anti-CD3, anti-CD8, and HEVlow HEVhigh anti-CD20 antibodies. E, the density þ þ 1,500 of CD3 T cells, CD8 T cells, and P < 0.001 CD20þ B cells is significantly higher 2 1,200 in HEVhigh than in HEVlow breast 900 tumors. The line in the center of each box represents the median value of CD20 600 the distribution, and the upper and CD20/mm 300 lower ends of the box are the upper 0 and lower quartiles, respectively. HEVlow HEVhigh Original magnification (A, 1.25 ; B, 10;C,10; and D, 5). 1,500

2 P < 0.001 1,200 CD8 900 600 CD8/mm 300 0 HEVlow HEVhigh

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G72 and G152, 2 HEV-specific antibodies (25) which recognize quantified the microvessel density of breast tumor sections the 6-sulfosialyl Lewis X ligands for lymphocyte l -selectin stained with antibodies against the pan-vascular marker þ (Supplementary Fig. S2). Together, these data indicated that CD34. We found no correlation between HEVs and CD34 þ the MECA-79 vessels present within human solid tumors are blood vessels present within breast tumor stroma (Fig. 2C), phenotypically identical to HEVs from lymphoid tissues and indicating that differences in the density of tumor HEVs are we, therefore, designated these vessels as tumor HEVs. not related to differences in tumor angiogenesis. Because HEVs are specifically located within lymphocyte- Density of tumor HEVs predicts T- and B-lymphocyte rich tumor areas, we then asked whether the density of tumor infiltration into breast tumors HEVs might correlate with lymphocyte infiltration. We quan- þ þ þ To define the functional consequence of tumor HEVs, we tified CD3 T cells, CD8 T cells, and CD20 B cells using focused on a retrospective cohort of 146 primary, invasive, optical grading and automatic cell count of tumor sections of nonmetastatic breast cancer patients operated at the ICR our breast cancer retrospective cohort (Supplementary between 1997 and 1998. Patient characteristics are described Fig. S3). We observed a strong correlation between the density þ þ in Supplementary Table S1. Absolute numbers of MECA-79 of tumor HEVs and tumor-infiltrating CD3 T cells (r ¼ 0.71; þ þ vessels present within the tumor area were quantified and the P < 0.001), CD8 T cells (r ¼ 0.66; P < 0.001), and CD20 B cells density of tumor HEVs (HEV/mm2) was calculated for each (r ¼ 0.73; P < 0.001; Fig. 2D and E; Supplementary Table S4). þ patient (Fig. 2A). The number and the density of MECA-79 These results were confirmed with a prospective cohort of 42 HEVs within breast tumor stroma were heterogeneous among breast cancer patients (Supplementary Fig. S4), indicating that different patients (Fig. 2B). Tumors with the highest density of tumor HEVs were associated with high levels of T-and B- HEVs were defined as HEVhigh (the highest tercile defined as lymphocyte infiltration in human breast tumors. cutoff) and tumors with the lowest density of HEVs were To further characterize the immune populations associated defined as HEVlow. Correlational analysis between the density with tumor HEVs, we conducted large-scale flow cytometric of HEVs and clinicopathologic parameters for these patients analyses on 30 freshly resected breast tumors. Tumors were revealed no statistical association between HEV density and classified into HEVhigh or HEVlow group according to the density þ tumor size, grade, nodal or hormonal receptor status, or of MECA-79 HEVs, quantified on adjacent paraffin-embedded adjuvant chemotherapy (Table 1). Aiming to examine poten- breast tumor sections. Immune populations and T-cell differ- tial links between HEV density and tumor angiogenesis, we entiation, activation, and functions were analyzed using 120 combinations of surface and intracellular markers (Fig. 3A; Supplementary Fig. S5). We observed a strong increase in the þ þ þ Table 1. Correlational analysis between the den- percentage of total CD4 and CD8 T cells and CD20 B cells in HEVhigh tumors as compared with HEVlow tumors (Fig. 3B). sity of tumor HEVs and clinicopathologic data þ þ þ Naive T cells (e.g., CCR7 , CD44 , and CD45RA CD62L ) and þ n (%) P central memory T cells (e.g., CD45RA CD62L ) that classically home toward secondary lymphoid organs through HEVs were Overall HEVlow HEVhigh greatly increased in HEVhigh tumors (Fig. 3B). In addition, population effector memory T cells (e.g., CD45RA CD62L ) and terminally þ differentiated T cells (e.g., TEMRA, CD45RA CD62L ), T cells n 97 49 expressing activation markers (e.g., CD69, CD25, HLA-DR, – – þ Range 0 0.189 0.197 10.658 CD86; refs. 5, 26), and cytotoxic CD8 T cells containing Median 0.009 0.614 granzyme A, granzyme B, and perforin were also present in Nodal status 0.52 larger proportion in HEVhigh breast tumors (Fig. 3B). Together, Negative 74 (50.7) 51 (52.6) 23 (46.9) these data indicate that tumor HEVs are associated with Positive 72 (49.3) 46 (47.4) 26 (53.1) increased numbers of both activated effector T cells and poorly Grade 0.22 differentiated T-cell populations, such as tumor-infiltrating – I II 76 (52.1) 54 (55.7) 22 (44.9) naive and central memory T cells. III 70 (47.9) 43 (44.3) 27 (55.1) Tumor size 0.45 Tumor HEVs correlate with T-helper 1 immune < 2 cm 71 (48.6) 45 (46.4) 26 (53.1) orientation 2 cm 75 (51.4) 52 (53.6) 23 (46.9) We then used quantitative real-time PCR to characterize ER status 0.75 the gene expression profile associated with the presence of Negative 25 (17.7) 16 (17.0) 9 (19.1) tumor HEVs in breast cancer. Expression levels of genes Positive 116 (82.3) 78 (83.0) 38 (80.9) related to lymphocyte migration, T-helper (TH) cell orienta- Missing 5 3 2 tion, cytotoxicity, and immune suppression were assessed in Chemotherapy 0.77 10 cryopreserved breast tumor tissues with a high density of No 53 (36.3) 36 (37.1) 17 (34.7) HEVs and 10 control tumors with a low density of HEVs, as Yes 93 (63.7) 61 (62.9) 32 (65.3) determined by immunohistochemistry (Fig. 4). Genes encod- CCL19 CCL21 CXCL13 Abbreviation: ER, estrogen receptor. ing lymphoid chemokines ( , , and ) and T-cell homing receptors (CCR7 and LSEL), which are

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HEVlow HEVhigh A B HEVlow HEVhigh 20 Figure 3. Large-scale flow *** Immune populations cytometric analysis of immune 15 *** populations associated with tumor HEVs in primary breast 10 *** *** cancer. A, 120 combinations of 5 *** surface and intracellular markers % of total cells were analyzed by flow cytometry 0 CD45+ CD3+ CD3+ CD3+ CD20+ for 30 breast tumors classified CD4+ CD8+ according to their density of HEVs high low T-cell differentiation (10 HEV vs. 20 HEV ). The 5 *** percentage of total cells from the *** 4 minimal (blue) to the maximal (red) *** level of expression was 3 *** *** determined for each patient and 2 *** plotted; gray areas represent analyses that were not done. B, a

% of total cells 1 high density of tumor HEVs is 0 associated with elevated numbers + + + + + + CD3 CD3 CD3 CD3 CD3 CD3 of different T-cell populations, + - + - 120 immune populations 120 immune CCR7 CD44 CD45RA CD45RA CD45RA-CD45RA+ including naive T cells, central + + CD62L CD62L CD62L- CD62L- memory T cells, effector memory 5 5 T-cell activation T-cell cytotoxicity T cells, activated T cells, and 4 *** 4 CD8þ T cells expressing cytotoxic *** 3 3 molecules. The mean (SD) *** percentage of total cells in HEVlow 2 2 *** *** (white bars) and HEVhigh (black *** * % of total cells 1 1 bars) breast tumors is shown for the different immune cell 0 0 P < P < + + + + CD8+ CD8+ CD8+ populations. *, 0.05; **, CD3 CD3 CD3 CD3 P < – + + + + + + 0.01; ***, 0.001; Mann Whitney -3.0 -2.8 -2.5 -2.3 -2.0 -1.8 -1.6 -1.3 -1.1 -0.8 -0.6 -0.4 -0.1 0.1 0.4 0.6 0.8 1.1 1.3 1.6 1.8 2.0 2.3 2.5 2.8 3.0 CD69 CD69 HLADR CD86 GZMB GZMA perf+ U test. CD25- CD25+

associated with naive and central memory T- and B-lympho- and YWHAZ). Transcripts of genes related to T-cell–cytotoxic cyte migration through HEVs, were greatly upregulated in granule components (PERF1, GZMA, GZMB, and GNLY) and high low HEV tumors as compared with HEV tumors. In contrast, TH1 orientation (IFNG and TBX21) were also statistically more no significant differences between 2 two groups of breast abundant in tumors with a high density of HEVs. In contrast, tumors were observed for housekeeping genes (HPRT, ACTB, the expression of genes associated with TH2(IL4 and GATA3)or

100 ** HEVlow HEVhigh ** *

(A.U.) (A.U.) ** ** ** ** ** ** ** 10 *

* *

Relative expressionRelative 1

Lymphocyte migration Control genes Cytotoxicity TH orientation Immune escape

Figure 4. Expression of genes related to lymphocyte migration, cytotoxicity, TH orientation, and immune escape determined in 20 breast tumors according to the density of tumor HEVs (10 HEVhigh vs. 10 HEVlow). Relative mRNA expression levels were adjusted to the level of GAPDH mRNA for each sample. The levels are represented as mean (SD) relative expression for HEVhigh (black bars) and HEVlow (white bars) tumors calculated by the DD cycling threshold (CT) method. *, P < 0.05; **, P < 0.01; Mann–Whitney U test.

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RORG IL17A TH17 ( and ) phenotypes, and that of genes related Density of tumor HEVs also showed a significant correla- to immune escape such as macrophage- (TGFB1, CD14, and tion with disease-free, metastasis-free, and overall survival CD68) and myeloid-derived suppressor cell (CD11b, CD33, and rates in multivariate analysis after adjustments using prog- COX2)-associated genes did not vary between HEVhigh and nostic factors previously identified (Table 2). The adjusted HEVlow tumors. Together, these results indicate that a high HRs of disease-free, metastasis-free, and overall survival rates density of HEVs in breast tumors is associated with the for patients with HEVlow tumors versus HEVhigh tumors were upregulation of genes involved in lymphocyte migration, TH1 2.68 (95% CI: 1.41–5.10; P ¼ 0.003), 3.28 (95% CI: 1.56–6.87; P ¼ adaptive immune response, and cytotoxic effector functions. 0.002), and 2.43 (95% CI: 1.16–5.06; P ¼ 0.018), respectively. Furthermore, we investigated the prognostic significance of þ Density of tumor HEVs predicts clinical outcome in tumor HEVs in the lymph node-positive group (N ) of breast þ breast cancer tumors (n ¼ 72). N patients with HEVhigh tumors had Tumor HEVs are associated with cytotoxic T-cell infiltra- significantly longer disease-free survival rate (P ¼ 0.02) and tion and TH1 immune orientation, previously identified as metastasis-free survival rate (P ¼ 0.03) than patients with critical for antitumor immunity both in human and mouse HEVlow tumors (Fig. 5B; Supplementary Table S5). Multivari- þ studies (1, 2); therefore, we next analyzed the clinical impact of ate analysis in N patients showed that a low density of tumor HEVs in our retrospective cohort of breast cancer patients. HEVs independently conferred a significantly higher risk of Univariate analysis indicated that patients with a high density relapse with adjusted HRs of 2.48 (95% CI: 1.16–5.30; P ¼ 0.019) of tumor HEVs had a significantly longer disease-free survival and 2.43 (95% CI: 1.09–5.40; P ¼ 0.029) for disease-free survival rate (P ¼ 0.01), metastasis-free survival (P ¼ 0.004), and overall and metastasis-free survival rates, respectively (Supplemen- survival (P ¼ 0.02) than patients with HEVlow tumors (Fig. 5A; tary Table S6). Absolute number of HEVs within tumor Supplementary Table S5). Using the "minimum P value" stroma, similar to HEV density, significantly predicted dis- approach, we verified that the density of tumor HEVs was ease-free and metastasis-free survival rates for both global and þ significantly correlated with metastasis-free survival rate for a N population of breast cancer patients (Supplementary large interval of cutoffs (Supplementary Fig. S6). Fig. S7).

A HEVlow HEVhigh B HEVlow HEVhigh

100 100

75 75

50 50

25 25 P = 0.01 P = 0.02 00 00 Disease-free survival (%) Disease-free survival (%) 240 7248 96 120 168144 240 7248 96 120 168144 Month Month Figure 5. The density of tumor HEVs predicts survival in breast 100 100 cancer. A and B, Kaplan–Meier curves for disease-free, 75 75 metastasis-free, and overall survival rates of 146 patients with 50 50 primary breast cancer (A) or 72 node-positive breast cancer (B) 25 25 patients, according to the density P = 0.004 P = 0.03 of tumor HEVs (HEVhigh, black line; 00 00 HEVlow, gray line). 240 7248 96 120 168144 Metastasis-free survival (%) 120 Metastasis-free survival (%) 240 7248 96 168144 Month Month 100 100

75 75

50 50

25 25 P = 0.02 P = 0.06 Overall survival (%) Overall

00 survival (%) Overall 00 240 7248 96 120 168144 240 7248 96 120 168144 Month Month

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Table 2. Multivariate analysis using the Cox proportional hazard model

Disease-free survival Overall survival

HR [95% CI] P (Wald) HR [95% CI] P (Wald)

HEV/mm2 Low 2.68 [1.41–5.10] 0.003 2.43 [1.16–5.06] 0.018 High 1 1 Tumor size <2cm 1 1 2 cm 3.44 [1.82–6.47] <0.001 3.08 [1.50–6.33] 0.002 Lymph node invasion N 11 Nþ 2.49 [1.31–4.70] 0.005 2.49 [1.19–5.23] 0.016 Grade SBR I–II 1 1 III 1.10 [0.62–1.98] 0.74 1.11 [0.59–2.07] 0.74 ER status Positive 1 1 Negative 1.33 [0.70–2.51] 0.34 1.70 [0.87–3.33] 0.12 Chemotherapy Yes 1 1 No 1.64 [0.86–3.13] 0.13 1.45 [0.69–3.02] 0.32

Abbreviation: ER, estrogen receptor.

Discussion These results reinforce the importance of better identifying blood vessel subtypes present within tumor stroma to better Lymphocyte migration into tumors remains poorly under- discriminate between their pro- and antitumor properties. stood, in spite of the critical impact of these cells on patients’ The favorable impact of effector memory T-cell and cyto- clinical outcomes (7–10). Our study shows, for the first time, toxic T-cell infiltration on patients’ clinical outcomes has that HEVs, blood vessels specialized in lymphocyte recruit- been showed in several types of cancer, including melanoma, ment, are frequently found in human solid tumors, predict colon, and ovarian carcinoma (5–8, 10, 11, 30, 31). The lymphocyte infiltration, immune orientation, and indepen- association between high densities of tumor HEVs and the dently correlate with favorable clinical outcome in both global presence of high numbers of these lymphocytes could, thus, þ and N populations of breast cancer patients. explain the prognosis value of HEVs in breast cancer. In Our results suggest that vascular specialization plays a addition, recruitment of naive and central memory T cells critical role in the control of lymphocyte infiltration into through the specific expression of sulfated l -selectin ligands tumors. Tumor HEVs are structurally and phenotypically simi- on tumor HEVs may contribute to the clinical impact of HEVs. lar to HEVs from patients suffering from autoimmune or Indeed, these undifferentiated populations of T cells, that have chronic inflammatory diseases such as rheumatoid arthritis, the ability to undergo self-renewal, have been shown to inflammatory bowel diseases, and autoimmune thyroiditis provide superior, long-term, antitumor response (32, 33). (15, 18, 19). The presence of HEVs in these pathologies has We can speculate that recruitment of naive T cells through been shown to participate in the development and the main- tumor HEVs and the generation, in the tumor vicinity, of high tenance of chronic inflammation through the recruitment of numbers of long-term, tumor antigen–specific central mem- large numbers of lymphocytes (27, 28). In contrast, HEVs have ory T cells and effector memory T cells with a TH1 orientation never been observed in normal nonlymphoid tissues. Therefore, capable of re-circulating throughout the body, may limit the HEVs may represent a local vascular response facilitating the establishment of metastasis in distant organs. In agreement rapid transfer of lymphocytes into both chronically inflamed with this hypothesis, mouse tumor models that stimulate tissues and tumor tissues. recruitment of naive T cells and priming at the tumor site We observed that HEV density within breast tumor stroma have been shown to promote both primary tumor and distant was not correlated with microvessel density. In addition, metastasis regression (34, 35). Furthermore, high densities of whereas microvessel density in breast tumors correlated with tumor HEVs were associated with B-lymphocyte infiltration poor clinical outcome in breast cancer (29), a high density of within tumor tissues. Activated B lymphocytes have been HEVs within tumor stroma was significantly associated with shown to critically impact T-cell activation and polarization longer disease-free survival rate of breast cancer patients. through antigen presentation, costimulatory molecule

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HEVs, Lymphocyte Infiltration, and Survival in Breast Cancer

expression, and cytokine production (36). Recruitment of B Disclosure of Potential Conflicts of Interest lymphocytes through tumor HEVs could, thus, be critical for efficient antitumor T-cell responses. No potential conflicts of interest were disclosed. Recent studies have established that the immune system actively participates in the clinical efficacy of conventional Authors' Contributions antitumor treatments such as chemotherapy and radiotherapy (3, 4). These observations give a rationale for therapeutic L. Martinet and I. Garrido designed research, carried out research, and manipulation of immune components. Nonetheless, immune- analyzed data; T. Filleron, S. Le Guellec, and P. Rochaix carried out research and analyzed data; E. Bellard carried out research; J-J. Fournie based therapies tested so far have obtained limited results (37), analyzed data; and J-P. Girard designed research, analyzed data, and wrote mainly because of the inability of effector immune cells to reach the manuscript. tumor tissues. Several mechanisms that restrict lymphocyte migration into tumors have previously been proposed (13, 14). Acknowledgements In contrast, our study is the first to describe a mechanism which facilitates lymphocyte infiltration into tumors—the presence of We thank Profs. Jean-Pierre Armand, Denis Querleu, Jean-Jacques Voigt, HEVs within the tumor microenvironment. By their ability to Henri Roche, Gilles Favre, and Francois¸ Amalric for their support. We are grateful to members of the Anatomopathology Service and the Biological recruit large numbers of circulating lymphocytes, tumor HEVs Resource Center (CRB) of the Institut Claudius Regaud, Toulouse, France, may represent new attractive targets for both cancer diagnosis for their help. This article is dedicated to the memory of Annie Raucoules and therapy. It will be important in future studies to further ("Tatou"). define the influence of tumor HEVs on clinical outcome, and to identify potential links between the density of tumor HEVs and Grant Support the response of patients to chemotherapy. Although HEV endothelial cells exhibit remarkable plasti- This work was supported by grants from the Ligue Nationale contre le Cancer (Equipe Labellisee Ligue 2009 to J-P. Girard and postdoctoral fellowship city and rapidly lose their specialized characteristics outside to L. Martinet), Region Midi-Pyrenees, and Foundation RITC. their natural tissue microenvironment (38), the factors and The costs of publication of this article were defrayed in part by the mechanisms involved in the induction and maintenance of payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate HEVs remain poorly understood. A better understanding of this fact. these factors may provide new opportunities to increase lymphocyte infiltration into tumors and enhance antitumor Received February 6, 2011; revised April 8, 2011; accepted April 11, 2011; immune mechanisms. published OnlineFirst August 16, 2011.

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OF10 Cancer Res; 71(17) September 1, 2011 Cancer Research

Human Solid Tumors Contain High Endothelial Venules: Association with T- and B-Lymphocyte Infiltration and Favorable Prognosis in Breast Cancer

Ludovic Martinet, Ignacio Garrido, Thomas Filleron, et al.

Cancer Res Published OnlineFirst August 16, 2011.

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