Occurrence of Tertiary Lymphoid Tissue Is Associated with T-Cell Inﬁltration and Predicts Better Prognosis in Early-Stage Colorectal Cancers

Published OnlineFirst February 12, 2014; DOI: 10.1158/1078-0432.CCR-13-2590

Clinical Cancer Imaging, Diagnosis, Prognosis Research See related article by Taube, p. 2023

Giuseppe Di Caro1, Francesca Bergomas1, Fabio Grizzi1, Andrea Doni1, Paolo Bianchi3, Alberto Malesci2, Luigi Laghi2,3, Paola Allavena1, Alberto Mantovani1,4, and Federica Marchesi1,4

Abstract Purpose: Tumor-infiltrating T lymphocytes (TIL) play a key role in the clinical outcome of human colorectal cancer; however, the dynamics of their recruitment along colorectal cancer clinical progression have not been fully elucidated. Tertiary lymphoid tissue (TLT) is an ectopic organized lymph node–like structure that typically forms at sites of chronic inflammation and is involved in adaptive immune responses. Its occurrence in cancer is sporadically documented and its role and clinical relevance is largely unknown. Experimental Design: The occurrence of TLT, the correlation with TILs, and the clinical relevance were evaluated retrospectively, in a cohort study involving a consecutive series of 351 patients with stage II and III colorectal cancer. The role of TLT in lymphocyte recruitment was assessed in a preclinical model of colorectal cancer. Results: In both human colorectal cancer and in a murine model of colorectal cancer, we identified organized TLT, highly vascularized (including high endothelial venules), and correlated with the density of þ CD3 TILs. Intravenous injection in mice of GFP splenocytes resulted in homing of lymphocytes to TLT, suggesting an active role of TLT in the recruitment of lymphocytes to tumor areas. Accordingly, TLT density and TIL infiltration correlated and were coordinated in predicting better patient’s outcome among patients with stage II colorectal cancer. Conclusions: We provide evidence that TLT is associated with lymphocyte infiltration in colorectal cancer, providing a pathway of recruitment for TILs. TLT cooperates with TILs in a coordinated antitumor immune response, when identifying patients with low-risk early-stage colorectal cancer, thus, representing a novel prognostic biomarker for colorectal cancer. Clin Cancer Res; 20(8); 2147–58. 2014 AACR.

Introduction adaptive immune response plays an active role in control- Immune infiltration is a fundamental component of ling cancer growth and dissemination. In fact, T-cell infil- solid tumors (1), its involvement in cancer progression tration correlates with favorable prognosis in various being documented by preclinical and clinical studies. The common neoplastic diseases, including colorectal cancer (2–5), emerging as a potential immune biomarker of outcome and promising therapeutic target. In colorectal Authors' Affiliations: Departments of 1Immunology and Inflammation; cancer, tumor-infiltrating lymphocytes (TILs) are associated and 2Gastroenterology; 3Laboratory of Molecular Gastroenterology, with favorable prognosis (2–4, 6–8), independently by 4 Humanitas Clinical and Research Center, Rozzano; and Department tumor–node–metastasis (TNM) stage (3, 6, 8), or only in of Biotechnologies and Translational Medicine, University of Milan, Milan, Italy stage II colorectal cancer (4). Surprisingly, despite the documented association between TILs and the clinical Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). outcome, the dynamics of T-cell recruitment and activation along colorectal cancer progression have not been fully G. Di Caro and F. Bergomas contributed equally to this work. elucidated. Corresponding Author: Federica Marchesi, Department of Biotech- Tertiary (or ectopic) lymphoid tissue (TLT) is a vascular- nologies and Translational Medicine, University of Milan—Humanitas Clinical and Research Center, Via Manzoni 56 20089 Rozzano, MI. Phone: ized immune compartment that forms at sites of exacerbat- 39-028-224-5113; Fax: 39-028-224-5101; E-mail: ed inflammatory reactions and is potentially involved in [email protected] the promotion of adaptive immune responses (9, 10). doi: 10.1158/1078-0432.CCR-13-2590 Similarly to secondary lymphoid organs, the function of 2014 American Association for Cancer Research. TLT is dependent on its structural organization, with

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to lymphoid tissues and lymph nodes (34). The pre- Translational Relevance sence of HEV in ectopic tissues indicates the pos- Our findings reveal that tertiary lymphoid tissue sibility for na€ve and central memory T lymphocytes to (TLT) is associated with lymphocyte infiltration in be recruited, thanks to their expression of L-selectin colorectal cancer, contributing to tumor-infiltrating (CD62L) and its specific binding to peripheral node lymphocyte (TIL) recruitment; TLT cooperates with addressin (PNAd), selectively expressed on HEV (35). TILs in a coordinated antitumor immune response In fact, the formation of HEV has been suggested to med- and in predicting better outcome in patient, thus, iate rapid recruitment of lymphocytes into chronically representing a novel prognostic biomarker for human inflamed tissues, including some tumor tissues (36). colorectal cancer. Improved understanding of the In the present study, we investigated the contribution of þ complexity of immune infiltration along the progres- TLT to CD3 T-cell infiltration in colorectal cancer. In a sion of colorectal cancer and the identification of retrospective cohort study, we identified highly organized immune biomarkers might help in the design of tai- lymphocyte aggregates representing bona fide TLT and we þ lored immune-based therapeutic approaches, targeting defined its prognostic relevance with respect to CD3 T-cell theimmunesystemaccordingtothestageofdisease. infiltration and demographics, clinical, and histopatholog- The organized accumulation of lymphocytes into ic variables and their interactions. In a preclinical model of ectopic lymphoid tissue in colorectal cancer is a pos- colitis-associated colorectal cancer, we functionally itive prognostic factor and may then represent a target addressed the question whether TLT is involved in the for therapeutic intervention in stage II colorectal can- recruitment of T lymphocytes into colon cancer tissue. A cer, with the aim to stimulate the immune system quantitative analysis, not subjected to estimation of pathol- against tumor progression. ogist has not been performed to date for lymphoid tissue in colorectal cancer. Thus, a clinically relevant definition of TLT in human colorectal cancer is expected to have important implications in the standardized assessment of lym- B-cell follicles, T-cell areas, and specialized hematic and phocyte infiltration in human colorectal cancer and in the lymphatic vessels, suggesting the possibility that it behaves design of clinical trials aimed to test novel immunothera- as a functional immune site (11, 12). TLT is found in the peutic approaches. inflamed area of several autoimmune diseases (13), chronic inflammatory conditions (14–16), and some tumor types (17–21). In these clinical settings, TLT improves the adap- Patients and Methods tive immune response to persisting antigens expressed in Patients the target organ (9, 10). In solid tumors, a role for TLT Tissue specimens from 351 stage II and III patients in the organization of the local immune response and in without any sign of metastatic disease at diagnosis who lymphocyte recruitment has been suggested (22), but solid consecutively underwent radical surgical resection for pT3 evidence has not been provided yet (23, 24). In human or pT4 colorectal cancer, were retrieved from the previous colorectal cancer, discrete aggregates of lymphocytes at the series (4). Patients’ demographics, clinical, and histopath- tumor invasive margins have been described and referred to ologic data were available and obtained from the Institu- as Crohn’s-like reaction (25–27); however, its functional tional Intranet; please refer to Supplementary Table S1 for role and the connection with the dispersed lymphocytic the list of the variables assessed. The absence of metastasis at infiltrate with prognostic value, also in regard to the type of diagnosis was assessed in all patients by combining histo- genetic instability, is surprisingly unexplored. Being a par- pathologic findings, surgical records, and perioperative adigm for the complex relationship between chronic imaging. To study the prediction of disease recurrences inflammation, T-cell–mediated immunity, and cancer pro- according to the state of immune infiltration, patients with gression (28, 29), colorectal cancer, represents an ideal pT1 or pT2 colorectal cancer, who have a very low risk of clinical setting to define the association between ectopic progression at diagnosis, and patients with perioperatively lymphoid tissue, a frequent manifestation of chronic detected metastases were excluded. To exclude potential inflammatory conditions, and infiltration of T cells with confounders in the study design, stage I patients were not antitumor properties. enrolled in the cohort studied for their unlikely occurrence As in secondary lymphoid organs, the formation of of disease recurrence, whereas patients with stage IV colo- TLT relies on few molecular mediators released by resi- rectal cancer were not included because they are character- dent stromal cells, including members of the lympho- ized by the presence of distant metastasis, which is an toxin family (30) and lymphorganogenic chemokines outcome event of our analysis. Patients who underwent (CXCL13 and CCL21), responsible for lymphoid cell neoadjuvant radiotherapy for rectal cancer were excluded recruitment (30–33). During the process of adult lym- from the study, because of the possibility of interference phoid neogenesis, similar molecular mediators and cel- with the assessment of the local immune response. Che- lular interactions induce formation of specialized vessels, motherapy treatment was administered and allocated by a including high endothelial venules (HEV) and lympha- nonrandom assignment according to adjuvant protocols in tics, which support traffic of lymphocytes from the blood use at the time of surgery.

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Study design the study, and written informed consent was obtained by Tissue specimens of patientswithcolorectalcancer the referring physician, at the time of surgery by each who consecutively underwent radical surgical resection patient. Slides were digitized using a computer-aided for pT3 or pT4 colorectal cancer at the Humanitas Clini- image analysis system (Olympus dotSlide). cal and Research Center (Rozzano, Milan, Italy) from TLT exhibited a distinct structural organization in colo- þ January 1997 to November 2005 were retrospectively rectal cancer, outlined by an area composed of CD3 cells studied. Investigators who were blinded to the results of and a compartment of CD3-negative lymphoid cells (B the morphologic analysis assembled a clinical retrospec- cells). To quantify TLT, an expert pathologist, who was tive database by collecting demographics, clinical, and blinded to any patient clinical data, randomly selected three histopathologic data from the institutional intranet (Sup- noncontiguous microscopic areas located at the tumor plementary Table S1). These variables, together with invasive front occupied by TLT. Computer-assisted mea- the median values of TLT and TIL IRA (immunoreactive surement of the selected areas was obtained as the percent- area), were tested as predictors of the outcome of patient. age ratio between TLT area and the total digitized tissue The outcome of patients who undergo radical resection surface. For each histologic section, the mean values of colorectal cancer is a variable affected by an event obtained in three different regions were calculated and used þ defined as any local tumor recurrences or any metachro- for the subsequent statistical analysis. CD3 TIL IRA was nous distant organ metastases and named disease-free quantified as previously described (4). Median values of the survival (DFS). To detect or exclude any postsurgical overall distribution of TLT and TIL IRA (2.68% and 2.06%, tumor recurrences, patients underwent thoracoabdom- respectively) were chosen as representative cutoff to per- inal computed tomography, abdominal ultrasonography, form statistical analyses. and chest radiography, which were done according to common protocols for surveillance. The observation peri- Statistical analysis od started immediately after the surgical procedure. The The association between the extent of TLT density and þ mean follow-up period of the cohort studied was 4.71 CD3 TILs, baseline characteristics and tumor features of years (SD ¼ 2.63 years) for DFS. The detection of tumor patient was estimated by Pearson simple linear regression recurrence or death was computed from diagnosis until analysis. A Cox proportional hazards model was devel- data were censored on May 30, 2010. To further assess oped to assess the role of TLT density and other demo- any possible biases, interaction analyses in predicting the graphic, clinical, and histopathologic features, in predict- prognosis of patient were performed for all the variables ing the occurrence of disease-free–specific survival. Time assessed to detect any effect modifier (P < 0.10). to follow-up was stopped at the time of the death of patient for any case unrelated to colorectal cancer disease, Immunohistochemistry and microsatellite status and this case was not considered an event of outcome. To From each patient enrolled in the study, 2-mmthick assess for confounders, COX multivariate analysis was tissue slided from formalin-processed and paraffin- performed by entering only variables and their significant embedded tumor sections were processed for immuno- interactions with a P value less than 0.20 at univariate histochemistry. After deparaffinization and rehydration, analysis. Interactions between variables were calculated sections were immersed in an antigen retrieval bath, by analyzing their multiplicative term in the Cox model. incubated with 3% H2O2 for 15 minutes. Slides were By a backward stepwise elimination approach, nonsig- autostained (IntelliPATH FLX; Biocare Medical) with pri- nificant variables, and their nonsignificant interactions, mary antibodies raised against CD3 (clone F7.2.38; were removed from the model. Interacting variables at Dako), CD20 (clone L26; Dako), PNAd (MECA-79; BD multivariate analysis (P < 0.10) were then tested for Pharmingen), Lyve-1 (ab14917; Abcam), CD21 (clone subgroups analysis accordingly. Differences in median EP3093; Abcam), a-smooth muscle actin (a-SMA) (clone values of TLT density between subsets of colorectal cancer 1A4; R&D Systems), and CXCL13 and CCL21 (AF801 and and DFS were tested by the Mann–Whitney U test and by AF457; R&D Systems). A 30-minute incubation with the the Cuzick trend test. Kaplan–Meier curves of DFS were DAKO Envision system (Dako) or the Anti-Goat Polymer plotted, whereas the log-rank test was used to compare Kit (BioCare) followed. Diaminobenzidine tetrahy- the curves of each subgroup of patients with colorectal drochloride (Dako) was used as chromogen. Nuclei were cancer. For each test, only two-sided P values lower than lightly counterstained with a freshly made hematoxylin 0.05 were considered statistically significant. All the anal- solution (Medite). Presence of fibrosis was assessed on 2- yses were done using Epi Info (Version 3.4.3), StatsDirect mm thick sections stained for 20 minutes with 0.1% Sirius Statistical software (Version 2.5), and GraphPad Prism red in saturated picric acid (Sigma-Aldrich). The sections software (Version 4.1). were further washed in water, mounted, and analyzed under an optical microscopy. Microsatellite status was Quantification of HEV in human colorectal cancer screened preliminarily for all cancers included in the Consecutive tumor slides from 20 patients with colo- studybytestinginstabilityatmononucleotiderepeats, rectal cancer were stained with antibodies raised against as previously described (37, 38). The Ethics Committee of PNAd.Foreachtumorslide,theabsolutenumbersof the Humanitas Clinical and Research Center approved PNAd-positive vessels within each intratumoral follicle

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and follicles associated with the normal mucosa were evidence of distant organ metastasis at diagnosis, by stain- quantified. ing with an anti-CD3–specific antibody and quantifying the area of TLT by computer-assisted image analysis. We Mice and murine models of colorectal cancer quantified TLT at the invasive front of the tumor, which Eight-week-old C57BL/6J and mice were purchased represents the tumor–host interface (Fig. 1A). Notably, in from Charles River Laboratories; eGFP/C57Bl/6 mice from the cohort of 351 patients with colorectal cancer, whole- The Jackson Laboratory. Procedures involving animals tissue visualization of CD3 infiltration showed a higher þ and their care were conformed to institutional guidelines density of TLT in tumors containing high density of CD3 in compliance with national and international laws and TILs (Fig. 2A, left), compared with tumors containing low þ policies. Mice were housed in a specific pathogen-free ani- density of CD3 TILs (Fig. 2A, right). In fact, the density of mal facility of the IRCCS Humanitas Clinical and Research TLT linearly correlated with the density of dispersed tumor- þ Center in individually ventilated cages. In the AOM/DSS infiltrating CD3 T cells (R ¼ 0.32; P < 0.001; Supplemen- (azoxymethane/dextran sodium sulphate) model, mice tary Table S2; Fig. 2B). þ developed adenomas as a result of the combined treat- Because lymphoid tissue contained PNAd HEVs, spe- ment with the carcinogen AOM (10 mg/kg; Sigma-Aldrich) cialized vessels with a key role in the traffic of T cells (Fig. and sequential administration of the mucosal irritant 1D), we aimed to explain the correlation of TLT and TILs in DSS [3 rounds of 2% DSS (MW ¼ 36,000–50,000; MP human colorectal cancer, by quantifying the distribution of Biomedicals)] in the drinking water. Adenomas develop HEV in colorectal cancer specimens. HEVs were present in the distal colon overtime during the different treatments mostly in the context of TLT and very rarely found in the and are evaluated after 10 weeks. surrounding tumor tissue. Comparison between lymphoid Additional details are included in the Supplementary tissue associated with the normal mucosa (Fig. 2C, left) and Methods. TLT at the invasive front of the tumor (Fig. 2C, right) showed an increased number of HEV associated with lym- Results phoid follicles in the tumor compartment (P < 0.05; Fig. Human colorectal cancer contains aggregates of 2D), indicating that the process of lymphoid neogenesis in T cells with features of TLT human colorectal cancer includes formation of HEV, which The lymphocytic reaction in colorectal cancer tissues might allow recruitment of T cells. includes dispersed TILs and discrete lymphoid aggregates, the latter referred to as Crohn-like reaction (25–27), which Density of TLT associates with a better prognosis in has not been characterized yet. By staining colon cancer patients with colorectal cancer specimens with an anti-CD3 antibody, we identified aggre- We then investigated the clinical significance of TLT in gates of lymphocytes displaying a distinct structural orga- relationship with TILs in a retrospective cohort study. Con- nization, compared with generic clusters of TILs (Fig. 1A). sidering the overall cohort, TLT IRA% at the invasive tumor These organized structures had features of TLT, with com- front ranged from 0% to 23.98%, with a median value of partmentalized T (Fig. 1B, left) and B (Fig. 1B, middle) areas, 2.68% (second–third quartiles, 0.75%–5.99%). TLT was þ sometimes with germinal centers, and a network of CD21 present in 276 (78.6%) of 351 tumors and the distribution follicular dendritic cells (FDC; Fig. 1B, right). Notably, TLT was skewed toward low values. Distributions of TLT accord- was often localized at the invasive front of the tumor, in ing to the patient histopathologic characteristics are stromal regions containing a considerable amount of cells described in Supplementary Table S2. We recorded 84 expressing a-SMA (Fig. 1C, left), a marker of activated events of colorectal cancer disease relapse (DFS) in 351 fibroblast cells usually associated with local inflammato- patients with stage II and III colorectal cancer. ry/fibrotic response. The lymphorganogenic chemokines Unadjusted univariate analysis showed that high TLT CCL21 (Fig. 1C, middle) and CXCL13 (Fig. 1C, right) were density ( median) significantly correlated with better out- also found inside lymphoid tissue, suggesting an active come [HR, 0.62; 95% confidence interval (CI), 0.40–0.97; recruitment of T and B cells and plasticity of these structures. Supplementary Table S3]. Importantly, to control for con- Staining of collagen fibers confirmed the fibrosis surround- founders, we performed multivariate Cox analysis that þ ing TLT (Fig. 1D, left). TLT contained PNAd HEV (Fig. 1D, revealed an interaction between higher densities middle) and Lyve-1–positive lymphatic vessels (Fig. 1D, ( median) of both TLT and TILs with nodal status in ¼ ¼ right), thus, confirming that lymphoid aggregates in human predicting patients relapse (P 0.07 and P 0.03, respec- colorectal cancer have features of TLT. tively), which suggests that the ability of TLT and TILs to predict patients relapse may change according to nodal Density of TLT in human colorectal cancer correlates status (Supplementary Table S3). Therefore, we performed with increased density of TILs and associates with a subgroup analysis revealing that in patients with node- increased number of HEVs negative colorectal cancer (n ¼ 185), a high density of TLT To analyze the relationship between TLT and TILs in (median, 2.68%) and of TILs (median, 2.06%) was human colorectal cancer, we systematically evaluated the associated with better prognosis compared with patients density of TLT in 351 tissue specimens from deeply invad- with a low density of TLT and TILs (log-rank test; P ¼ 0.02 ing (pT3/pT4) patients with colorectal cancer without and P ¼ 0.02, respectively; Fig. 3A and B, left); conversely,

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Figure 1. Characterization of aggregates of T cells with features of TLT in human colorectal cancer. A, a representative image obtained from a virtual digital slide of a colorectal tumor specimen. Staining with anti-CD3 antibody allows identification of the T- lymphocytic reaction, including the presence of lymphoid aggregates at the invasive front (asterisks). On the right are magnifications of the lymphoid aggregates. B, lymph node–like follicles (dotted lines) in human colorectal cancer specimens are þ composed of CD3 T cells (left), þ CD20 B cells (middle), and þ networks of CD21 FDCs (right). Sections in left and middle are consecutive sections and show the compartmentalization between B and T cells in the follicle. C, intratumor lymphoid tissue is prevalently associated with tumor stroma, containing–- a-SMA-positive cells (left). The lymphoid chemokines CCL21 (middle) and CXCL13 (right) are present inside lymphoid follicles. D, staining with Sirius red evidenced the presence of collagen fibers closed to vascular structures (asterisks; left). Vessels within lymphoid tissue þ include PNAd HEV (middle) as þ well as Lyve-1 lymphatic vessels (right), suggesting traffic of naïve and memory lymphocytes. Sections in D middle and right are consecutive sections, showing HEV and lymphatic vessels in the same follicle. Dot lines indicate follicle contour. Scale bars, 1 mm (A); 200 mm(B and C, left; D, left and right); and 100 mm (C, middle and right; D, middle).

TLT and TIL densities were irrelevant to predict the prog- (no relapse) vs. N1 (relapse; P ¼ 0.43); N2 (no relapse) vs. nosis of patients with node-positive colorectal cancer (n ¼ N2 (relapse; P ¼ 0.15)], thus, behaving as a prognostic 166; log-rank test; P ¼ 0.46 and P ¼ 0.64, respectively; Fig. biomarker only in early-stage patients (Fig. 3C, left). 3A and B, right). Moreover, we further conﬁrmed that the To address the question whether the immune response prognostic behavior of TLT and TILs varies with disease comprising of TLT and TILs has no impact at all in stage III extent in a different analysis, performed with continuous colorectal cancer, we performed a stage-by-stage analysis, values. Similarly to TILs (Fig. 3C, right), among patients showing the ability of nodal status to identify relapses of with node-negative colorectal cancer, TLT density was sig- patient with colorectal cancer among subgroups of TLT niﬁcantly lower in patients who relapsed (n ¼ 26) than in density (Table 1). Similarly to TILs, nodal involvement was patients with no evidence of disease recurrence [n ¼ 159; N0 associated with worst outcome in subgroups of patients (no relapse) vs. N0 (relapse; P ¼ 0.03; Fig. 3C, left)]. with colorectal cancer with intermediate TLT density Conversely, TLT density did not differ in nodal-positive (0.1%–4.2%, P ¼ 0.001; 4.2%–8.4%, P ¼ 0.05) but not in patients (N1 and N2) with or without tumor recurrence [N1 patients with colorectal cancer with very low or very high

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AB 30 P < 0.001 r = 0.32 20

10 TLT density (%) TLT

CD3 CD3 0 0102030 CDCD3+ TIL density (%) 60 * 50

HEV number/TLT 10 PNAd PNAd 0 Normal Tumor

Figure 2. Correlation of tumor-associated TLT with increased density of T cells and formation of HEVs in human colorectal cancer. A, representative images obtained from virtual digital slides of two colorectal tumor specimens with a low (left) and high (right) density of infiltrating T cells and TLT. Staining with anti-CD3 antibody allows identification of lymphoid tissue and infiltrating lymphocytes. Asterisks, lymphoid tissue. B, correlation þ between CD3 TILs (% IRA) and TLT density (% IRA) in 351 patients with colorectal cancer. A P value by Pearson simple linear regression analysis. C, increased number of HEV associated with TLT in the tumor compartment. Comparison between lymphoid tissue associated with the normal mucosa (left) and TLT at the invasive front of the tumor (right). Quantification of HEV in tumor lymphoid tissue compared with lymphoid follicles associated with normal mucosa. HEV were quantified by computer-assisted image quantitative analysis on slides from 20 paraffin-embedded cancer tissue specimens stained with anti-PNAd antibody. Bars, number of HEV in each follicle (D). Two-tailed P values by the t test. , P < 0.05. Scale bars, 1 mm (A); and 200 mm(C).

immune values (0%, P ¼ 0.16; 8.32%, P ¼ 0.33; Table 1). progression. Figure 3D showed that TLT correlates with Consequently, only very low and very high TLT densities TILs density (median) only in patients who did not (0%; 8.32%) were associated with better outcome in both experience relapse (P ¼ 0.001), but not in those who stage II (P ¼ 0.04) or stage III (P ¼ 0.02) colorectal cancer relapsed (P ¼ 0.28; Fig. 3D), thus, suggesting that the two and might, therefore, operate as stage-independent predic- biomarkers are coordinated in mediating the antitumor tors of survival (Table 1). response only among patients with a good prognosis. These results suggest that the prognostic behavior of TLT Our data have shown that microsatellite instable (MSI) is similar to that of TILs and prompted us to test which one patients with colorectal cancer had a tendency to better was the best prognostic marker. However, because these prognosis compared with those with microsatellite stable biomarkers identified overlapping populations, which were (MSS) phenotype, although not statistically significant (HR, mutually dependent in predicting prognosis (data not 0.56; 95% CI, 0.27–1.16; P ¼ 0.12; Supplementary Table shown), we developed two stepwise, backward Cox multi- S3). To further address whether the prognostic value of MSI variate analysis models (model A and model B), to test might differ according to the density of TLT, we performed the independency of their prognostic performance, with Kaplan–Meier curves with subgroups analyses (Supplemen- respect to other demographics, clinical, histopathologic tary Fig. S1). The analysis showed that MSI colorectal cancer features, and microsatellite status in stage II colorectal was not significantly associated with prognosis in both cancer (Table 2). Model A and model B revealed that higher patients with TLT density low (

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Figure 3. Clinical relevance of TLT in 351 patients with colorectal cancer. A and B, Kaplan–Meier curves showing DFS, according to TLT and TIL density. A high density of TLT (median, 2.68%) and of TILs (median, 2,06%) is associated with better outcome in patients with node-negative colorectal cancer (P ¼ 0.02 and P ¼ 0.02, respectively, n ¼ 185; A and B, left), but not in those with node-positive colorectal cancer (P ¼ 0.46 and P ¼ 0.64, respectively, n ¼ 166; A and B, right). C and D, coordination of TLT and TIL immune inﬁltration. The prognostic behavior of TLT (C, left) and TILs (C, right) varies with disease extent. Among patients with node-negative colorectal cancer, TLT density is signiﬁcantly lower in patients who relapsed (n ¼ 26) than in patients with no evidence of disease recurrence (n ¼ 159; P ¼ 0.03), whereas it does not differ in nodal-positive þ patients with or without tumor recurrence. Distribution of TLT IRA% according to the CD3 density (median) in relapsing and not relapsing patients with stage II colorectal cancer. TLT and TILs correlate only among patients that do not relapse (D). P (C, left): N0 (no relapse) versus N0 (relapse) P ¼ 0.03; N1 (no relapse) versus N1 (relapse) P ¼ 0.43; N2 (no relapse) versus N2 (relapse) P ¼ 0.15. P (C, right): N0 (no relapse) versus N0 (relapse) P ¼ 0.01; N1 (no relapse) versus N1 (relapse) P ¼ 0.74; N2 (no relapse) versus N2 (relapse) P ¼ 0.51.

þ the dynamics of CD3 T-cell recruitment and activation driven colon carcinogenesis, lymphoid tissue signi- at the tumor site have not been clarified. The coordina- ficantly increased compared with control mice (Sup- tion between TLT and TILs in predicting prognosis in plementary Fig. S2D), consistently with the local induc- human colorectal cancer prompted us to better clarify tion of TLT in chronically inflamed tissues. To define the the association of TLT and T-cell infiltration. We took association of TLT with T lymphocytes in colorectal þ advantage of a preclinical model of inflammation-driven cancer, we compared CD3 TcellswithinTLTofcon- carcinogenesis (AOM/DSS; ref. 39), which would reca- trol and AOM/DSS mice (Fig. 4A, left and middle). pitulate the formation of TLT associated with chronic Quantification of the T-cell infiltrate indicated that þ inflammatory conditions (23). Organized accumulations the number of CD3 T cells in lymphoid tissue signi- of lymphoid cells are present in murine colon mucosa ficantly increased in AOM/DSS mice (Fig. 4A, right). of AOM/DSS mice, both adjacent to the normal crypts Thus, as evidenced by our previous clinical analysis, this (Supplementary Fig. S2A, left) and in the tumor region result further confirmed in a preclinical model that (Supplementary Fig. S2A, right). The aggregates are com- TLT associates with increased T-cell infiltration in colo- prised of mostly B lymphocytes and include an area of rectal cancer. T cells (Supplementary Fig. S2B) and a network of FDCs To test the hypothesis that TLT is actively involved (Supplementary Fig. S2C, left). The lymphoid chemokine in the recruitment of lymphocytes, we intravenously þ CXCL13 is expressed at high levels in the aggregates injected GFP splenocytes into control mice and mice (Supplementary Fig. S2C, right), consistent with the subjectedtotheAOM/DSSprotocol.After24hours, þ predominant presence of B cells. During inflammation- GFP cells localized in TLT of AOM/DSS mice, whereas

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Table 1. Prediction of risk for disease relapse by nodal status in 351 pT3/pT4 colorectal cancer

Relapse Cox hazards model No Yes (n ¼ 267) (n ¼ 84) HR (95% CI) P CD3 TILs density <1% N0 32 11 1.00 ref. N1–N2 33 17 1.39 (0.65–2.97) 0.39 1%–5% N0 77 11 1.00 ref. N1–N2 52 25 2.91 (1.43–5.91) 0.003 5%–10% N0 32 2 1.00 ref. N1–N2 15 15 12.12 (2.75–53.38) 0.001 10% N0 18 2 1.00 ref. N1–N2 8 1 1.12 (0.10–12.36) 0.92 TLT density No TLT N0 25 8 1.00 ref. N1–N2 25 17 1.81 (0.78–4.20) 0.16 0%–4.16% N0 62 12 1.00 ref. N1–N2 45 31 2.90 (1.48–5.65) 0.001 4.16%–8.32% N0 42 4 1.00 ref. N1–N2 20 7 3.27 (0.95–11.17) 0.05 8.32% N0 30 2 1.00 ref. N1–N2 18 3 2.41 (0.40–14.45) 0.33

NOTE: Colorectal cancer were subgrouped according to the density of CD3þ TILs and TLT at the tumor invasive front. Abbreviations: N0, no lymph node involvement; N1, one to three nodes involved; N2, more than or equal to four nodes involved.

þ very few or none were observed in lymphoid tissue of of GFP cells in colorectal cancer–associated lymphoid control mice (Fig. 4B, left and middle). Whole-tissue tissue (Fig. 4B, right), thus, conﬁrming that TLT in the analysis evidenced a signiﬁcant increase in the density tumor mediates recruitment of lymphocytes.

Table 2. The multivariate Cox hazard model for predictive factors of disease relapse in 185 stage II pT3/pT4 colorectal cancer Multivariate analysis

Relapse Model A Model B

No Yes (n ¼ 159) (n ¼ 26) HR (95% CI) P HR (95% CI) P TLT density

NOTE: Multivariate analysis was performed by introducing TLT density (median) in model A and TIL density (median) in model B and by entering all other variables with a P value less than 0.20 at univariate analysis. By a backward stepwise elimination approach, nonsigniﬁcant variables, and their nonsigniﬁcant interactions, were removed from the model. apT3, invading through the muscularis propria into subserosa or into nonperitonealized pericolic or perirectal tissues. pT4, directly invading adjacent organs or perforating visceral peritoneum. bVariants mucinous or medullary.

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Figure 4. TLT mediates increased lymphocyte infiltration in a preclinical model of colorectal cancer. A, expansion of TLT associates with increased number of þ þ CD3 T cells. Staining with anti-CD3 antibody in lymphoid tissue (dotted circle) of control mice (left) and AOMDSS mice (middle). Quantification of CD3 cells in colon þ tissues of mice was performed by computer-assisted image analysis and indicates increased number of CD3 T cells in the TLT of AOMDSS mice, þ compared with control mice (right). B, TLT is actively involved in the recruitment of lymphocytes. GFP splenocytes were intravenously injected in control and AOM/ þ DSS mice and whole colons analyzed by immunofluorescence after 24 hours. Representative images of GFP cells in a lymphoid follicle (dotted circle) of control (left) þ þ and AOM/DSS (middle) mice. Quantification of GFP cells was performed on 20-mm thick sections from the whole colon; Each dot represents the density of GFP cells in the TLT analyzed (right). One representative of two experiments performed (n ¼ 3 mice; Ctrl; n ¼ 5 mice AOM/DSS; bars, SEM). C, the vessel network draining þ þ þ lymphoid tissue (B220 B cells) in the colon mucosa of control (left) and tumor-bearing mice (middle) includes CD31 blood vessels and Lyve1 lymphatic vessels. Morphometric analysis on colon whole mounts indicates vessel expansion around and inside lymphoid tissue of AOMDSS mice (right; n ¼ 6 mice; Ctrl; n ¼ 6 mice þ AOM/DSS; bars, SEM). D, TLT contains functional HEVs. Staining with and anti-PNAd antibody indicates presence of HEV in lymphoid tissue (left). CD3 T þ lymphocytes circulate into PNAd HEV, and localize in closeproximity tothevessel wall (asterisks;middle).Thenumber of HEVincreases inlymphoid tissue of tumor- bearing mice (right). Scale bar, 200 mm(A–C); 100 mm(D,left);50mm (D, middle). Two-tailed P values by the t test. , P < 0.005; , P < 0.001.

þ Whole-mount tissue analysis of TLT in murine colons 1 lymphatic vessels (Fig. 4C, left and middle). Quanti- allowed to visualize a dense network of vessels surround- ﬁcation of the volume of vessels draining TLT evidenced a þ ing TLT, which included CD31 blood vessels and Lyve- signiﬁcant increase in AOM/DSS mice compared with

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þ control mice (Fig. 4C, right). Among CD31 vessels, Our stage-by-stage analysis showed that only very high þ lymphoid tissue contained PNAd HEVs (Fig. 4D, left); or very low TLT densities have prognostic impact in stage þ CD3 T cells were localized inside HEV and in close III and behave as independent predictors of survival. proximity to the vessel wall (Fig. 4D, middle). The num- Essentially, although the antitumor impact of immune þ ber of PNAd HEV within lymphoid tissue of AOM/DSS infiltration is more relevant in stage II colorectal cancer, mice was higher compared with control mice (Fig. 4D, only the strongest immune responses have an impact on right), thus, confirming that TLT formation in colon the prognosis in stage III. However, the number of cancer associates with expansion of a vascular network patients with colorectal cancer identified by very high and can sustain lymphocyte recruitment. cutoff values is proportionally scarce and thus, limits its clinical impact. The generation of threshold values and combined immune values is a critical issue in the assess- Discussion ment of the prognostic abilities of immune cells and The recognition of the key role of tumor-infiltrating should be carefully managed. In previous studies, the leukocytes in cancer has triggered the efforts toward a better use of these cutoffs together with combined values definition of the complexity of the immune response in obtained from markers identifying overlapping immune tumors, with the relevant clinical perspective to identify cell populations has led to claim the futility of pathologic novel prognostic biomarkers, which might help in the staging (3, 8). This strategy fostered statistical analysis but design of immune-based therapeutic approaches. In this identified a very small benchmarking population of þ scenario, CD3 lymphocytes have recently emerged as a patients with colorectal cancer devoid of TILs and with robust immune biomarker in several solid tumors, includ- a dismal prognosis (3, 8), with limited clinical prognostic þ ing colorectal cancer. In our study, we show that CD3 T- relevance when addressing surveillance strategies in the infiltrating lymphocytes at the tumor invasive front of overall population of colorectal cancer. colorectal cancer can localize in organized aggregates, com- The type of genomic instability has been proposed as an prised of T- and B-cell areas and a network of FDCs, thus important variable to be included in the design of studies on with features of TLT, in which clinical relevance in relation- immune cells and prognosis (40). MSI patients with colo- ship to TILs has been so far unexplored. rectal cancer have a better prognosis over MSS (41, 42) Intratumor TLT was associated with an increased density together with a higher lymphocytic reaction at the tumor of TILs and to a dense vascular network, including HEV and site (4, 6, 26, 37). Our data further addressed this issue by lymphatic vessels, required to ensure proper traffic of lym- showing that the prognostic value of TLT density is inde- phocytes within lymphoid organs, and thus, suggesting that pendent by MSI, thus, not being influenced by their lower TLT has the capability to sustain traffic of T cells. The metastatic potential. presence of the chemokines CCL19 and CXCL13 within Protocol variability for quantification of immune cells, TLT strongly supports the hypothesis that lymphoid tissue is together with inconsistent statistical design is a critical relevant to mediate active recruitment of lymphocytes into factor contributing to discrepancy of results among studies. the tumor, which was then confirmed by intravenous injec- Importantly, although the assessment of TILs, irregularly tion of GFP splenocytes in mice. and heterogeneously dispersed within the tissue is chal- The clinical relevance of tumor-infiltrating T cells in lenging, TLT are easy to detect under an optical microscope human colorectal cancer has been extensively documented and by image analysis, being organized as cellular aggre- (2–6, 8, 26, 27). Previous analyses of T-cell infiltration in gates. A worldwide concerted action (Immunoscore) is þ patients with colorectal cancer claimed CD3 TILs as better ongoing, aimed at assessing the actual clinical usefulness indicator of prognosis than TNM tumor staging (3). How- of a standardized methodologic assessment of T-cell infil- þ ever, on accurate analysis, CD3 TILs retained prognostic tration (43, 44). The results presented here further suggest significance only in patients with nodal-negative colorectal that the quantification of organized TLT is a feasible and þ cancer (4), thus, identifying CD3 TILS as a prognostic easy approach to this issue in the context of TNM staging biomarker for stage II colorectal cancer and suggesting the and that TLT and TILs are coordinated in their clinical possibility to implement the TNM-based system with one relevance in early-stage human colorectal cancer. Thus, TLT þ developed upon the densities of CD3 TILs. About TLT, assessment should also be considered when the prognostic here, we took advantage of a computer-assisted image value of TILs is investigated. analysis, which is objective and statistically more relevant, We found that TLT and TILs populations are highly providing continuous distributions of immune cell densi- overlapping in their extent and prognostic abilities ties. By these means, we showed for the first time that, in because these biomarkers were mutually dependent in accordance with TILs, TLT are not predominant over tumor predicting the prognosis of patient. However, we provid- staging in predicting patient outcome and their prognostic ed phenomenologic evidence that the antitumor algo- relevance varies with the state of disease progression at rithm represented by TLT and TIL densities is coordinated diagnosis. Thus, they may contribute to tumor control at only when identifying colorectal cancer patients who the early stages of the disease, while they progressively loose were not relapsing. Thus, it is conceivable to hypothesize this capability along with tumor progression and occur- that T-cell recruitment and the mounting of an efficient rence of lymph node metastasis. T-cell antitumor immune reaction is favored by the

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presence of a local immune environment like TLT at the responsiveness of novel immunotherapeutic strategies in tumor site, whereas the pathways of activation of these colorectal cancer. two immune players seem to differ among colorectal cancers with an aggressive behavior. According to their Disclosure of Potential Conflicts of Interest cellular composition, rich in T and B cells, their structural No potential conflicts of interest were disclosed. organization and their intratumor localization, it is rea- sonable to hypothesize that TLT at the invasive front in Disclaimer human colorectal cancer might collect T cells in close The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article. proximity to cancer cells, potentially improving the efficiency of the antitumor response. Authors' Contributions B cells are a relevant component of TLT. In human cancer, Conception and design: A. Malesci, L. Laghi, F. Marchesi B cells are known to promote tumor immunity by several Development of methodology: G. Di Caro, F. Bergomas, F. Grizzi, A. Doni mechanisms, including production of antibodies directed Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G. Di Caro, F. Bergomas, F. Grizzi, A. Malesci to tumor-specific antigens, antigen presentation, and Analysis and interpretation of data (e.g., statistical analysis, biosta- enhancement of T-cell antitumor activity (45, 46) all func- tistics, computational analysis): G. Di Caro, F. Grizzi, P. Bianchi, L. Laghi, tions being highly favored by the presence of an immune F. Marchesi Writing, review, and/or revision of the manuscript: G. Di Caro, F. Grizzi, site. In this regard, our data suggest that also a humoral P. Bianchi, L. Laghi, P. Allavena, A. Mantovani, F. Marchesi immune response organized at the tumor site, within TLT, Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): A. Malesci might be a player in the generation of an antitumor immune Study supervision: A. Malesci, A. Mantovani, F. Marchesi response with prognostic relevance. Overall, the occurrence and modulation of TLT may be Acknowledgments particularly significant in the development and the respon- The authors thank Chiara Rossi. siveness of novel immunotherapeutic approaches. In this scenario, we also provided further phenomenologic evi- Grant Support dence to the idea that nodal invasion is crucial in deter- This work was supported by the Italian Association for Cancer Research (AIRC) Italy (grant number MFAG-11677; to F. Marchesi and IG-12051; to P. mining the efficiency and the coordination of adaptive Allavena) and the Italian Ministry of University and Research, FIRB grant antitumor responses (47). Nevertheless, the lack of murine (RBAP11H2R9; to A. Mantovani). G. Di Caro is supported by a fellowship models properly reproducing the progression of colorectal from Fondazione Umberto Veronesi. The costs of publication of this article were defrayed in part by the cancer across its stages of disease might explain at least in payment of page charges. This article must therefore be hereby marked part recent failures in translating immunotherapeutic advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate approaches to clinical practice. Therefore, we suggest that this fact. TNM stage of disease should be a critical variable in the Received September 24, 2013; revised December 17, 2013; accepted design and the assessment of clinical trials aimed to test the January 12, 2014; published OnlineFirst February 12, 2014.

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2158 Clin Cancer Res; 20(8) April 15, 2014 Clinical Cancer Research

Occurrence of Tertiary Lymphoid Tissue Is Associated with T-Cell Infiltration and Predicts Better Prognosis in Early-Stage Colorectal Cancers

Giuseppe Di Caro, Francesca Bergomas, Fabio Grizzi, et al.

Clin Cancer Res 2014;20:2147-2158. Published OnlineFirst February 12, 2014.

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