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    Combined antiangiogenic and anti-PD-L1 therapy stimulates tumor immunity through HEV formation. Allen Elizabeth,Jabouille Arnaud,Rivera Lee B,Lodewijckx Inge,Missiaen Rindert,Steri Veronica,Feyen Kevin,Tawney Jaime,Hanahan Douglas,Michael Iacovos P,Bergers Gabriele Science translational medicine Inhibitors of VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) are commonly used in the clinic, but their beneficial effects are only observed in a subset of patients and limited by induction of diverse relapse mechanisms. We describe the up-regulation of an adaptive immunosuppressive pathway during antiangiogenic therapy, by which PD-L1 (programmed cell death ligand 1), the ligand of the negative immune checkpoint regulator PD-1 (programmed cell death protein 1), is enhanced by interferon-γ-expressing T cells in distinct intratumoral cell types in refractory pancreatic, breast, and brain tumor mouse models. Successful treatment with a combination of anti-VEGFR2 and anti-PD-L1 antibodies induced high endothelial venules (HEVs) in PyMT (polyoma middle T oncoprotein) breast cancer and RT2-PNET (Rip1-Tag2 pancreatic neuroendocrine tumors), but not in glioblastoma (GBM). These HEVs promoted lymphocyte infiltration and activity through activation of lymphotoxin β receptor (LTβR) signaling. Further activation of LTβR signaling in tumor vessels using an agonistic antibody enhanced HEV formation, immunity, and subsequent apoptosis and necrosis in pancreatic and mammary tumors. Finally, LTβR agonists induced HEVs in recalcitrant GBM, enhanced cytotoxic T cell (CTL) activity, and thereby sensitized tumors to antiangiogenic/anti-PD-L1 therapy. Together, our preclinical studies provide evidence that anti-PD-L1 therapy can sensitize tumors to antiangiogenic therapy and prolong its efficacy, and conversely, antiangiogenic therapy can improve anti-PD-L1 treatment specifically when it generates intratumoral HEVs that facilitate enhanced CTL infiltration, activity, and tumor cell destruction. 10.1126/scitranslmed.aak9679
    Multicenter phase II trial of Camrelizumab combined with Apatinib and Eribulin in heavily pretreated patients with advanced triple-negative breast cancer. Nature communications In the later-line setting or for patients with PD-L1-negative tumors, immunotherapy-based regimens remain ineffective against advanced triple-negative breast cancer (TNBC). In this multicentered phase II trial (NCT04303741), 46 patients with pretreated advanced TNBC were enrolled to receive camrelizumab 200 mg (day 1), and apatinib 250 mg daily, plus eribulin 1.4 mg/m (day 1 and 8) on a 21-day cycle until progression, or unacceptable toxicity. Primary endpoint was objective response rate (ORR) according to RECIST 1.1. Secondary endpoints included toxicities, disease control rate (DCR), clinical benefit rate, progression-free survival (PFS), and 1-year overall survival. With a median of 3 lines of prior chemotherapy in the advanced setting, 17.4% had received PD-1/PD-L1 blockade plus chemotherapy for advanced disease. The ORR was 37.0% (17/46, 95% CI 23.2-52.5). The DCR was 87.0% (40/46, 95% CI 73.7-95.1). Median PFS was 8.1 (95% CI 4.6-10.3) months. Tertiary lymphoid structure was associated with higher ORR. Patients with lower tumor PML or PLOD3 expression had favorable ORR and PFS. PD-L1 status was not associated with ORR/PFS. Grade 3/4 treatment-related adverse events occurred in 19 (41.3%) of 46 patients. Camrelizumab plus apatinib and eribulin shows promising efficacy with a measurable safety profile in patients with heavily pretreated advanced TNBC. 10.1038/s41467-022-30569-0
    Expression of the MHC class II in triple-negative breast cancer is associated with tumor-infiltrating lymphocytes and interferon signaling. Park In Ah,Hwang Seong-Hye,Song In Hye,Heo Sun-Hee,Kim Young-Ae,Bang Won Seon,Park Hye Seon,Lee Miseon,Gong Gyungyub,Lee Hee Jin PloS one Tumor-infiltrating lymphocytes (TILs) have been known for their strong prognostic and predictive significance in triple-negative breast cancer (TNBC). Several mechanisms for TIL influx in TNBC have been elucidated. Major histocompatibility complex class II (MHC-II) is an essential component of the adaptive immune system and is generally restricted to the surface of antigen-presenting cells. However, it has been reported that interferon-gamma signaling may induce MHC-II in almost all cell types, including those derived from cancer. We aimed to examine the relationship between MHC-II expression in tumor cells and the amount of TILs in 681 patients with TNBC. Further, the prognostic significance of MHC-II and the association of MHC-II with a couple of molecules involved in the interferon signaling pathway were investigated using immunohistochemical staining. Higher MHC-II expression in tumor cells was associated with the absence of lymphovascular invasion (p = 0.042); larger amounts of TILs (p < 0.001); frequent formations of tertiary lymphoid structures (p < 0.001); higher expression of myxovirus resistance gene A, one of the main mediators of the interferon signaling pathway (p < 0.001); and higher expression of double-stranded RNA-activated protein kinase, which can be induced by interferons (p = 0.008). Moreover, tumors that showed high MHC class I expression and any positivity for MHC-II had larger amounts of CD4- and CD8-positive T lymphocytes (p < 0.001). Positive MHC-II expression in tumor cells was associated with better disease-free survival in patients who had lymph node metastasis (p = 0.009). In conclusion, MHC-II expression in tumor cells was closely associated with an increase in TIL number and interferon signaling in TNBC. Further studies are warranted to improve our understanding regarding TIL influx, as well as patients' responses to immunotherapy. 10.1371/journal.pone.0182786
    CXCL13-producing TFH cells link immune suppression and adaptive memory in human breast cancer. Gu-Trantien Chunyan,Migliori Edoardo,Buisseret Laurence,de Wind Alexandre,Brohée Sylvain,Garaud Soizic,Noël Grégory,Dang Chi Vu Luan,Lodewyckx Jean-Nicolas,Naveaux Céline,Duvillier Hugues,Goriely Stanislas,Larsimont Denis,Willard-Gallo Karen JCI insight T follicular helper cells (TFH cells) are important regulators of antigen-specific B cell responses. The B cell chemoattractant CXCL13 has recently been linked with TFH cell infiltration and improved survival in human cancer. Although human TFH cells can produce CXCL13, their immune functions are currently unknown. This study presents data from human breast cancer, advocating a role for tumor-infiltrating CXCL13-producing (CXCR5-) TFH cells, here named TFHX13 cells, in promoting local memory B cell differentiation. TFHX13 cells potentially trigger tertiary lymphoid structure formation and thereby generate germinal center B cell responses at the tumor site. Follicular DCs are not potent CXCL13 producers in breast tumor tissues. We used the TFH cell markers PD-1 and ICOS to identify distinct effector and regulatory CD4+ T cell subpopulations in breast tumors. TFHX13 cells are an important component of the PD-1hiICOSint effector subpopulation and coexpanded with PD-1intICOShiFOXP3hi Tregs. IL2 deprivation induces CXCL13 expression in vitro with a synergistic effect from TGFβ1, providing insight into TFHX13 cell differentiation in response to Treg accumulation, similar to conventional TFH cell responses. Our data suggest that human TFHX13 cell differentiation may be a key factor in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses in the chronic inflammatory breast cancer microenvironment. 10.1172/jci.insight.91487
    Tertiary lymphoid structures are associated with higher tumor grade in primary operable breast cancer patients. Figenschau Stine L,Fismen Silje,Fenton Kristin A,Fenton Christopher,Mortensen Elin S BMC cancer BACKGROUND:Tertiary lymphoid structures (TLS) are highly organized immune cell aggregates that develop at sites of inflammation or infection in non-lymphoid organs. Despite the described role of inflammation in tumor progression, it is still unclear whether the process of lymphoid neogenesis and biological function of ectopic lymphoid tissue in tumors are beneficial or detrimental to tumor growth. In this study we analysed if TLS are found in human breast carcinomas and its association with clinicopathological parameters. METHODS:In a patient group (n = 290) who underwent primary surgery between 2011 and 2012 we assessed the interrelationship between the presence of TLS in breast tumors and clinicopathological factors. Prognostic factors were entered into a binary logistic regression model for identifying independent predictors for intratumoral TLS formation. RESULTS:There was a positive association between the grade of immune cell infiltration within the tumor and important prognostic parameters such as hormone receptor status, tumor grade and lymph node involvement. The majority of patients with high grade infiltration of immune cells had TLS positive tumors. In addition to the degree of immune cell infiltration, the presence of TLS was associated with organized immune cell aggregates, hormone receptor status and tumor grade. Tumors with histological grade 3 were the strongest predictor for the presence of TLS in a multivariate regression model. The model also predicted that the odds for having intratumoral TLS formation were ten times higher for patients with high grade of inflammation than low grade. CONCLUSIONS:Human breast carcinomas frequently contain TLS and the presence of these structures is associated with aggressive forms of tumors. Locally generated immune response with potentially antitumor immunity may control tumorigenesis and metastasis. Thus, defining the role of TLS formation in breast carcinomas may lead to alternative therapeutic approaches targeting the immune system. 10.1186/s12885-015-1116-1
    Relationship between Tertiary Lymphoid Structure and the Prognosis and Clinicopathologic Characteristics in Solid Tumors. Zhao Zhan,Ding Hui,Lin Zheng-Bin,Qiu Sheng-Hui,Zhang Yi-Ran,Guo Yan-Guan,Chu Xiao-Dong,Sam Loi I,Pan Jing-Hua,Pan Yun-Long International journal of medical sciences An increasing number of studies had shown that tertiary lymphoid structure (TLS) plays an important role in tumor progression. However, the prognostic role of TLS in various tumors remains controversial. This meta-analysis aims to investigate the clinicopathological and prognostic values of TLS in solid tumors. A systematic search was conducted in PubMed, EMBASE and Cochrane Library undated to November 2, 2020. Odds ratios of clinical parameters, hazard ratio (HR) of overall survival (OS), relapse-free survival (RFS), disease-free survival (DFS) and relapse rate were calculated in order to evaluate the relationship between TLS expression and clinicopathological or prognostic values in different tumors. 27 eligible studies including 6647 patients with different types of tumors were analyzed. High TLS expression was associated with a longer OS (HR = 0.66, 95% CI: 0.50 - 0.86, = 0.002) and RFS (HR = 0.61, 95% CI: 0.47 - 0.79, = 0.0001). Moreover, high TLS levels in tumor were associated with a low risk of recurrence (HR = 0.43, 95% CI: 0.32 - 0.57,  < 0.0001). However, there was no relationship between TLS expression and DFS. Meanwhile, high TLS expression was associated with smaller tumor size ( < 0.00001) and higher tumor infiltrating lymphocytes (TILs). Furthermore, the subgroup analysis showed high TLS expression that may be associated with a lower clinical grading and N stage in breast cancer and colorectal cancer. High TLS expression is associated with the longer OS and RFS in solid tumors, and a lower risk of cancer relapse. Meanwhile, high TLS expression is also associated with a smaller tumor size, higher infiltration of TILs, lower clinical grading and N stage in the tumor. Therefore, high TLS expression in the tumor is a favorable prognostic biomarker for solid tumor patients. 10.7150/ijms.56347
    Quantifying Tertiary Lymphoid Structure-Associated Genes in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissues. Gu-Trantien Chunyan,Garaud Soizic,Migliori Edoardo,Solinas Cinzia,Lodewyckx Jean-Nicolas,Willard-Gallo Karen Methods in molecular biology (Clifton, N.J.) Tertiary lymphoid structures (TLS) have been detected in several types of human solid tumors. These structures are thought to regulate local adaptive immune responses that can promote or antagonize tumor progression. Despite positive prognostic values associated with a TLS presence in several studies, discrepancies still exist. TLS are structurally organized entities composed of varying numbers of multiple cell types making their assessment in tumor tissues, particularly biopsies, challenging. Immunohistochemical staining of TLS-related cell populations is the most frequently used method for identifying and scoring them; however, TLS-related gene expression has also been explored. The protocols described are detailed to allow the user to quantify TLS-related gene expression on formalin-fixed paraffin-embedded human breast tumor tissues. 10.1007/978-1-4939-8709-2_9
    Immune infiltration in human tumors: a prognostic factor that should not be ignored. Pagès F,Galon J,Dieu-Nosjean M-C,Tartour E,Sautès-Fridman C,Fridman W-H Oncogene The natural history of a tumor includes phases of 'in situ' growth, invasion, extravasation and metastasis. During these phases, tumor cells interact with their microenvironment and are influenced by signals coming from stromal, endothelial, inflammatory and immune cells. Indeed, tumors are often infiltrated by various numbers of lymphocytes, macrophages or mast cells. It is generally believed that the latter produce factors that maintain chronic inflammation and promote tumor growth, whereas lymphocytes may control cancer outcome, as evidenced in mouse models. In this study, we analyze data from large cohorts of human tumors, clearly establishing that infiltration of the primary tumor by memory T cells, particularly of the Th1 and cytotoxic types, is the strongest prognostic factor in terms of freedom from disease and overall survival at all stages of clinical disease. We review data suggesting that tertiary lymphoid structures adjacent to tumors and composed of mature dendritic cells (T and B cells organized as germinal centers) may be the site of an antitumor reaction. We propose an immune scoring based on the type, density and location of lymphocyte infiltrates as a novel prognostic factor for use in addition to tumor node metastasis staging to predict disease-free survival and to aid in decisions regarding adjuvant therapies in early stage human cancers. 10.1038/onc.2009.416
    Spatial distribution of B cells and lymphocyte clusters as a predictor of triple-negative breast cancer outcome. Wortman Juliana C,He Ting-Fang,Solomon Shawn,Zhang Robert Z,Rosario Anthony,Wang Roger,Tu Travis Y,Schmolze Daniel,Yuan Yuan,Yost Susan E,Li Xuefei,Levine Herbert,Atwal Gurinder,Lee Peter P,Yu Clare C NPJ breast cancer While tumor infiltration by CD8 T cells is now widely accepted to predict outcomes, the clinical significance of intratumoral B cells is less clear. We hypothesized that spatial distribution rather than density of B cells within tumors may provide prognostic significance. We developed statistical techniques (fractal dimension differences and a box-counting method 'occupancy') to analyze the spatial distribution of tumor-infiltrating lymphocytes (TILs) in human triple-negative breast cancer (TNBC). Our results indicate that B cells in good outcome tumors (no recurrence within 5 years) are spatially dispersed, while B cells in poor outcome tumors (recurrence within 3 years) are more confined. While most TILs are located within the stroma, increased numbers of spatially dispersed lymphocytes within cancer cell islands are associated with a good prognosis. B cells and T cells often form lymphocyte clusters (LCs) identified via density-based clustering. LCs consist either of T cells only or heterotypic mixtures of B and T cells. Pure B cell LCs were negligible in number. Compared to tertiary lymphoid structures (TLS), LCs have fewer lymphocytes at lower densities. Both types of LCs are more abundant and more spatially dispersed in good outcomes compared to poor outcome tumors. Heterotypic LCs in good outcome tumors are smaller and more numerous compared to poor outcome. Heterotypic LCs are also closer to cancer islands in a good outcome, with LC size decreasing as they get closer to cancer cell islands. These results illuminate the significance of the spatial distribution of B cells and LCs within tumors. 10.1038/s41523-021-00291-z
    Immune Crosstalk Between Lymph Nodes and Breast Carcinomas, With a Focus on B Cells. Alberts Elena,Wall Isobelle,Calado Dinis Pedro,Grigoriadis Anita Frontiers in molecular biosciences Lymph nodes (LNs) are highly organized secondary lymphoid organs, and reflective of immune responses to infection, injuries, or the presence of cancer. Extensive molecular and morphological analyses of immune and stromal features in tumors and LNs of breast cancer patients have revealed novel patterns indicative of disease progression. Within LNs, there are dynamic structures called germinal centers (GCs), that act as the immunological hubs for B cell development and generation of affinity matured memory B and antibody-producing plasma cells. Acting as a bridge between systemic and local immunity, associations are observed between the frequency of GCs within cancer-free LNs, the levels of stromal tumor infiltrating lymphocytes, and cancer progression. Scattered throughout the tumor microenvironment (TME) or aggregated in clusters forming tertiary lymphoid structures (TLS), the occurrence of tumor infiltrating B cells (TIL-Bs) has been linked mostly to superior disease trajectories in solid cancers. Recent TIL-Bs profiling studies have revealed a plethora of different TIL-B populations, their functional roles, and whether they are derived from GC reactions in the LN, and/or locally from GC-like structures within the TME remains to be investigated. However, parallels between the immunogenic nature of LNs as a pre-metastatic niche, TIL-B populations within the TME, and the presence of TLS will help to decipher local and widespread TIL-Bs responses and their influence on cancer progression to the lymphatics. Therapies that enhance TIL-Bs responses in the LN GC and/or in GC-like structures in the TME are thus emerging management strategies for breast and other cancer patients. 10.3389/fmolb.2021.673051
    Tumor-infiltrating lymphocyte composition, organization and PD-1/ PD-L1 expression are linked in breast cancer. Buisseret Laurence,Garaud Soizic,de Wind Alexandre,Van den Eynden Gert,Boisson Anais,Solinas Cinzia,Gu-Trantien Chunyan,Naveaux Céline,Lodewyckx Jean-Nicolas,Duvillier Hugues,Craciun Ligia,Veys Isabelle,Larsimont Denis,Piccart-Gebhart Martine,Stagg John,Sotiriou Christos,Willard-Gallo Karen Oncoimmunology The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4 T cells and CD19 B cells and a lower mean frequency of CD8 T cells compare with normal tissues, increasing the CD4/CD8 T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4 T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies. 10.1080/2162402X.2016.1257452
    Fluorescent Multiplex Immunohistochemistry Coupled With Other State-Of-The-Art Techniques to Systematically Characterize the Tumor Immune Microenvironment. Boisson Anaïs,Noël Grégory,Saiselet Manuel,Rodrigues-Vitória Joël,Thomas Noémie,Fontsa Mireille Langouo,Sofronii Doïna,Naveaux Céline,Duvillier Hugues,Craciun Ligia,Larsimont Denis,Awada Ahmad,Detours Vincent,Willard-Gallo Karen,Garaud Soizic Frontiers in molecular biosciences Our expanding knowledge of the interactions between tumor cells and their microenvironment has helped to revolutionize cancer treatments, including the more recent development of immunotherapies. Immune cells are an important component of the tumor microenvironment that influence progression and treatment responses, particularly to the new immunotherapies. Technological advances that help to decipher the complexity and diversity of the tumor immune microenvironment (TIME) are increasingly used in translational research and biomarker studies. Current techniques that facilitate TIME evaluation include flow cytometry, multiplex bead-based immunoassays, chromogenic immunohistochemistry (IHC), fluorescent multiplex IHC, immunofluorescence, and spatial transcriptomics. This article offers an overview of our representative data, discusses the application of each approach to studies of the TIME, including their advantages and challenges, and reviews the potential clinical applications. Flow cytometry and chromogenic and fluorescent multiplex IHC were used to immune profile a HER2+ breast cancer, illustrating some points. Spatial transcriptomic analysis of a luminal B breast tumor demonstrated that important additional insight can be gained from this new technique. Finally, the development of a multiplex panel to identify proliferating B cells, Tfh, and Tfr cells on the same tissue section demonstrates their co-localization in tertiary lymphoid structures. 10.3389/fmolb.2021.673042
    Chemotherapy Coupled to Macrophage Inhibition Induces T-cell and B-cell Infiltration and Durable Regression in Triple-Negative Breast Cancer. Cancer research Immunosuppressive elements within the tumor microenvironment, such as tumor-associated macrophages (TAM), can present a barrier to successful antitumor responses by cytolytic T cells. Here we employed preclinical syngeneic p53 null mouse models of triple-negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small-molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was effective in treating several highly aggressive TNBC murine mammary tumor and lung metastasis models. Single-cell RNA sequencing characterized tumor-infiltrating lymphocytes including Th cells and antigen-presenting B cells that were highly enriched in responders to combination therapy. In one model that exhibited long-term posttreatment tumor regression, high-dimensional imaging techniques identified the close spatial localization of B220+/CD86+-activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks posttreatment. The transcriptional and metabolic heterogeneity of TAMs was also characterized in two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model was highly conserved in human claudin-low breast cancers, and high expression of the TAM signature correlated with reduced overall survival in patients with breast cancer. This TAM signature may help identify human patients with claudin-low breast cancer that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rational immunotherapy combinations. SIGNIFICANCE:Immunostimulatory chemotherapy combined with pharmacologic inhibition of TAMs results in durable treatment responses elicited by Th cells and B cells in claudin-low TNBC models. 10.1158/0008-5472.CAN-21-3714
    FOXP1 negatively regulates tumor infiltrating lymphocyte migration in human breast cancer. De Silva Pushpamali,Garaud Soizic,Solinas Cinzia,de Wind Alexandre,Van den Eyden Gert,Jose Vinu,Gu-Trantien Chunyan,Migliori Edoardo,Boisson Anaïs,Naveaux Céline,Duvillier Hugues,Craciun Ligia,Larsimont Denis,Piccart-Gebhart Martine,Willard-Gallo Karen EBioMedicine BACKGROUND:FOXP1, a transcriptional regulator of lymphocyte development, is abnormally expressed in some human tumors. This study investigated FOXP1-mediated regulation of tumor infiltrating lymphocytes (TIL) in untreated primary breast cancer (BC). METHODS:FOXP1 expression was analyzed in tissues from primary untreated breast tumors, BC cell lines and the METABRIC gene expression BC dataset. Cytokine and chemokine expression and lymphocyte migration in response to primary tumor supernatants (SN) was compared between FOXP1 and FOXP1 primary BC. FINDING:FOXP1 expression was higher in estrogen receptor positive compared to negative BC. FOXP1 tumors were significantly associated with lower TIL and fewer tertiary lymphoid structures (TLS) compared to FOXP1 BC. Silencing FOXP1 in BC cell lines positively impacted cytokine and chemokine expression with the inverse effect associated with overexpression. CXCL9, CXCL10, CXCL11, CXCL13, CX3CL, CCL20, IL2, IL21, GZMB and IFNG expression decreased while IL10 and TGFβ increased in FOXP1 compared to FOXP1 primary BC. Lymphocyte migration using primary BC supernatants detected decreased mobility toward FOXP1 supernatants. FOXP1 BC expresses higher levels of chemokines driving TIL migration. The METABRIC gene expression dataset analysis show FOXP1 expression is associated with unfavorable BC outcomes. INTERPRETATION:These data identify FOXP1 as an important negative regulator of immune responses in BC via its regulation of cytokine and chemokine expression. FUND: Belgian Fund for Scientific Research (FNRS 3.4513.12F) and Opération Télévie (7.4636.13F and 7.4609.15F), Fonds J.C. Heuson and Fonds Lambeau-Marteaux. 10.1016/j.ebiom.2018.11.066
    The balance between breast cancer and the immune system: Challenges for prognosis and clinical benefit from immunotherapies. Baxevanis Constantin N,Fortis Sotirios P,Perez Sonia A Seminars in cancer biology Cancer evolution is a complex process influenced by genetic factors and extracellular stimuli that trigger signaling pathways to coordinate the continuous and dynamic interaction between tumor cells and the elements of the immune system. For over 20 years now, the immune mechanisms controlling cancer progression have been the focus of intensive research. It is well established that the immune system conveys protective antitumor immunity by destroying immunogenic tumor variants, but also facilitates tumor progression by shaping tumor immunogenicity in a process called "immunoediting". It is also clear that immune-guided tumor editing is associated with tumor evasion from immune surveillance and therefore reinforcing the endogenous antitumor immunity is a desired goal in the context of cancer therapies. The tumor microenvironment (TME) is a complex network which consists of various cell types and factors having important roles regarding tumor development and progression. Tumor infiltrating lymphocytes (TILs) and other tumor infiltrating immune cells (TIICs) are key to our understanding of tumor immune surveillance based on tumor immunogenicity, whereby the densities and location of TILs and TIICs in the tumor regions, as well as their functional programs (comprising the "immunoscore") have a prominent role for prognosis and prediction for several cancers. The presence of tertiary lymphoid structures (TLS) in the TME or in peritumoral areas has an influence on the locally produced antitumor immune response, and therefore also has a significant prognostic impact. The cross-talk between elements of the immune system with tumor cells in the TME is greatly influenced by hypoxia, the gut and/or the local microbiota, and several metabolic elements, which, in a dynamic interplay, have a crucial role for tumor cell heterogeneity and reprogramming of immune cells along their activation and differentiation pathways. Taking into consideration the recent clinical success with the application immunotherapies for the treatment of several cancer types, increasing endeavors have been made to gain better insights into the mechanisms underlying phenotypic and metabolic profiles in the context of tumor progression and immunotherapy. In this review we will address (i) the role of TILs, TIICs and TLS in breast cancer (BCa); (ii) the different metabolic-based pathways used by immune and breast cancer cells; and (iii) implications for immunotherapy-based strategies in BCa. 10.1016/j.semcancer.2019.12.018
    B cells and tertiary lymphoid structures promote immunotherapy response. Nature Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity, although these have been less well-studied in ICB treatment. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets. 10.1038/s41586-019-1922-8
    ICAM1 expression is induced by proinflammatory cytokines and associated with TLS formation in aggressive breast cancer subtypes. Figenschau Stine L,Knutsen Erik,Urbarova Ilona,Fenton Christopher,Elston Bryan,Perander Maria,Mortensen Elin S,Fenton Kristin A Scientific reports Intratumoral formation of tertiary lymphoid structures (TLS) within the tumor microenvironment is considered to be a consequence of antigen challenge during anti-tumor responses. Intracellular adhesion molecule 1 (ICAM1) has been implicated in a variety of immune and inflammatory responses, in addition to associate with triple negative breast cancer (TNBC). In this study, we detected TLS in the aggressive tumor phenotypes TNBC, HER2+ and luminal B, whereas the TLS negative group contained solely tumors of the luminal A subtype. We show that ICAM1 is exclusively expressed in TNBC and HER2 enriched subtypes known to be associated with inflammation and the formation of TLS. Furthermore, cell from normal mammary epithelium and breast cancer cell lines expressed ICAM1 upon stimulation with the proinflammatory cytokines TNFα, IL1β and IFNγ. ICAM1 overexpression was induced in MCF7, MDA-MB-468 and SK-BR-3 cells regardless of hormone receptor status. Taken together, our findings show that ICAM1 is expressed in aggressive subtypes of breast cancer and its expression is inducible by well-known proinflammatory cytokines. ICAM1 may be an attractive molecular target for TNBC, but further investigations elucidating the role of ICAM1 in targeted therapies have to take into consideration selective subtypes of breast cancer. 10.1038/s41598-018-29604-2
    Immune Checkpoint Molecules on Tumor-Infiltrating Lymphocytes and Their Association with Tertiary Lymphoid Structures in Human Breast Cancer. Solinas Cinzia,Garaud Soizic,De Silva Pushpamali,Boisson Anaïs,Van den Eynden Gert,de Wind Alexandre,Risso Paolo,Rodrigues Vitória Joel,Richard François,Migliori Edoardo,Noël Grégory,Duvillier Hugues,Craciun Ligia,Veys Isabelle,Awada Ahmad,Detours Vincent,Larsimont Denis,Piccart-Gebhart Martine,Willard-Gallo Karen Frontiers in immunology There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4 or CD8 T cells and CTLA-4 is expressed on CD4 T cells. The global proportion of PD-L1, PD-L2, LAG3, and TIM3 tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1, PD-L2, LAG3, and TIM3 cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even within the same molecular subtype. These data indicate that assessing the levels of immune checkpoint molecule expression in an individual patient has important implications for the success of therapeutically targeting them in BC. 10.3389/fimmu.2017.01412
    Molecular Signature of Tumor-Associated High Endothelial Venules That Can Predict Breast Cancer Survival. Cancer immunology research High endothelial venules (HEV) are specialized post-capillary venules that recruit naïve lymphocytes to lymph nodes. HEVs are essential for the development of adaptive immunity. HEVs can also develop in tumors where they are thought to be important for recruiting naïve T cells and B cells into the tumors and locally enhancing antitumor immunity by supporting the formation of tertiary lymphoid structures. Herein, we used comparative transcriptome analysis of human breast cancer to investigate genes differentially expressed between tumor-associated HEVs and the rest of the tumor vasculature. Tumor vessels highly expressing HEV-upregulated genes, such as the homeobox gene MEOX2 and the tetraspanin gene TSPAN7, were associated with extensive infiltration of T and B cells and the occurrence of tertiary lymphoid structures, which is known to predict therapeutic responses to immune-checkpoint inhibitors. Moreover, high transcript counts of these genes in clinical tumor specimens were associated with a significant survival benefit in advanced breast cancer. The molecular signature of HEVs identified herein may be useful for guiding immunotherapies and provides a new direction for investigating tumor-associated HEVs and their clinical significance. See related Spotlight by Gallimore, p. 371. 10.1158/2326-6066.CIR-21-0369
    Tertiary lymphoid structures: prognostic significance and relationship with tumour-infiltrating lymphocytes in triple-negative breast cancer. Lee Hee Jin,Park In Ah,Song In Hye,Shin Su-Jin,Kim Joo Young,Yu Jong Han,Gong Gyungyub Journal of clinical pathology BACKGROUND:Tumour-infiltrating lymphocytes (TILs) have a strong prognostic significance, particularly in triple-negative breast cancer (TNBC). One important source of TILs in breast cancer is tertiary lymphoid structures (TLSs). OBJECTIVE:To carry out a histological analysis of surgically resected TNBC to identify the location of TLSs, the relationship between TLSs and TILs and their prognostic significance in TNBC. METHODS:We retrospectively analysed 769 patients with TNBC. RESULTS:TILs were defined as the percentage of stroma of invasive carcinoma infiltrated by lymphocytes. TLSs were mainly present within adjacent terminal duct lobular units and around in situ components. TNBC with higher levels of TILs showed a higher nuclear grade, lower lymphovascular invasion, less accompanying in situ component, a homogeneous growth pattern, necrosis in invasive areas, low levels of tumour stroma, high levels of peritumoral lymphocytic infiltration and moderate to abundant TLSs in adjacent tissue. TILs, the degree of peritumoral lymphocytic infiltration and adjacent TLSs were prognostic factors for disease-free and overall survival. Although the TIL level did not have a prognostic value in stage I, it added significant prognostic information for stages II and III. Conversely, patients with high levels of TILs did not show prognostic differences according to the pTNM stage. Patients with high levels of TILs (> 60%) and moderate to abundant TLSs had significantly better disease-free survival than those with high levels of TILs but none or few TLSs. CONCLUSIONS:TLSs are frequently present in TNBC and are closely associated with TILs. TILs provide additional prognostic information in patients with TNBC with a higher pTNM stage. 10.1136/jclinpath-2015-203089
    Prognostic impact of tertiary lymphoid structures in breast cancer prognosis: a systematic review and meta-analysis. Zhang Na-Na,Qu Feng-Jin,Liu Hao,Li Zhu-Jun,Zhang Yu-Chi,Han Xuan,Zhu Zi-Yu,Lv Yi Cancer cell international BACKGROUND:Tertiary lymphoid structures (TLSs), organizationally resemble lymph nodes, are frequently present in breast cancer (BCa). It is usually, but not always, associated with a positive prognosis or immunotherapy response in cancer patients. This meta-analysis was performed to assess the prognostic and clinical impact of TLSs in BCa. METHODS:We conducted a systematic search in PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, and WanFang Database to obtain eligible research data up to May 30, 2021. This meta-analysis is focusing on the studies evaluated the prognostic value of TLSs and the associated clinicopathologic indicators, related gene expression and survival. STATA software 16.0 software was used to assess the prognostic significance and clinical impact of TLSs. RESULTS:Nine studies involved with 2281 cases were incorporated in this meta-analysis, in which four of them evaluated the prognostic value of TLSs. There are 6 studies assessed the relationship of TLSs and 4 studies investigated the clinicopathologic parameters as well as the key gene expression, respectively. The results showed the presence of TLSs were predicting a better OS (HR = 0.61, 95% CI: 0.51-0.73, p < 0.001) and DFS (HR = 0.40, 95% CI: 0.17-0.93, p < 0.001) of BCa patients. It also revealed that the presence of TLSs was significantly correlated with tumor differentiation (p < 0.001), pTNM stage (p < 0.001), lymph node metastasis (p < 0.001), and TILs density (p < 0.001) of BCa, and the expression of Her2 (p < 0.001), ER (p < 0.001), PR (p < 0.001) and Ki67 (p = 0.009) of the tumor cell. CONCLUSION:Our results indicated that high levels of TLSs could predict a favorable prognosis for BCa. Moreover, the TLSs were significantly correlated with the clinicopathological indicators and the critical gene expression of BCa, indicating its potential clinical impact on BCa patients. 10.1186/s12935-021-02242-x
    The 12-CK Score: Global Measurement of Tertiary Lymphoid Structures. Li Roger,Berglund Anders,Zemp Logan,Dhillon Jasreman,Putney Ryan,Kim Youngchul,Jain Rohit K,Grass G Daniel,Conejo-Garcia José,Mulé James J Frontiers in immunology There is emerging evidence that the adaptive anti-tumor activity may be orchestrated by secondary lymphoid organ-like aggregates residing in the tumor microenvironment. Known as tertiary lymphoid structures, these lymphoid aggregates serve as key outposts for lymphocyte recruitment, priming and activation. They have been linked to favorable outcomes in many tumor types, and more recently, have been shown to be effective predictors of response to immune checkpoint blockade. We have previously described a 12-chemokine (12-CK) transcriptional score which recapitulates an overwhelming enrichment for immune-related and inflammation-related genes in colorectal carcinoma. Subsequently, the 12-CK score was found to prognosticate favorable survival in multiple tumors types including melanoma, breast cancer, and bladder cancer. In the current study, we summarize the discovery and validation of the 12-CK score in various tumor types, its relationship to TLSs found within the tumor microenvironment, and explore its potential role as both a prognostic and predictive marker in the treatment of various cancers. 10.3389/fimmu.2021.694079
    Hypoxia and anaerobic metabolism relate with immunologically cold breast cancer and poor prognosis. Breast cancer research and treatment PURPOSE:Hypoxia-Inducible Factor HIF1α and lactate dehydrogenase LDHA drive anaerobic tumor metabolism and define clinical aggressiveness. We investigated their expression in breast cancer and their role in immune response and prognosis of breast cancer. METHODS:Tissue material from 175 breast cancer patients treated in a prospective study were analyzed with immunohistochemistry for HIF1α and LDH5 expression, in parallel with the tumor-infiltrating lymphocyte TIL-density and tertiary lymphoid structure TLS-density. RESULTS:High LDH5 expression was noted in 48/175 tumors, and this was related to HIF1α overexpression (p < 0.0001), triple-negative TNBC histology (p = 0.01), poor disease-specific survival (p < 0.007), metastasis (p < 0.01), and locoregional recurrence (p = 0.03). High HIF1α expression, noted in 39/175 cases, was linked with low steroid receptor expression (p < 0.05), her2 overexpression (p = 0.01), poor survival (p < 0.04), and high metastasis rates (p < 0.004). High TIL-density in the invading tumor front (TILinv) was linked with low LDH5 and HIF expression (p < 0.0001) and better prognosis (p < 0.02). High TIL-density in inner tumor areas (TILinn) was significantly linked with TNBC. Multivariate analysis showed that PgR-status (p = 0.003, HR 2.99, 95% CI 1.4-6.0), TILinv (p = 0.02, HR 2.31, 95% CI 1.1-4.8), LDH5 (p = 0.01, HR 2.43, 95% CI 1.2-5.0), N-stage (p = 0.04, HR 2.42, 95% CI 1.0-5.8), T-stage (p = 0.04, HR 2.31, 95% CI 1.0-5.1), and her2 status (p = 0.05, HR 2.01, 95% CI 1.0-4.2) were independent variables defining death events. CONCLUSION:Overexpression of LDH5, an event directly related to HIF1α overexpression, characterizes a third of breast tumors, which is more frequent in TNBC. Both HIF1α and LDH5 define cold breast cancer microenvironment and poor prognosis. A rational is provided to study further whether metabolic manipulations targeting HIF and LDH5 may enhance the antitumor immune response in breast cancer. 10.1007/s10549-022-06609-0
    Tertiary lymphoid structures and associated plasma cells play an important role in the biology of triple-negative breast cancers. Seow Dominique Yuan Bin,Yeong Joe Poh Sheng,Lim Johnathan Xiande,Chia Noel,Lim Jeffrey Chun Tatt,Ong Clara Chong Hui,Tan Puay Hoon,Iqbal Jabed Breast cancer research and treatment PURPOSE:Triple-negative breast cancers (TNBC) are aggressive tumours that exhibit abundant lymphoid infiltrates which modulate tumour behaviour. Recent findings suggest that TNBC with higher densities of plasma cells are associated with a favourable prognosis, and tertiary lymphoid structures (TLS) have prognostic significance. Here, we studied the phenotype and function of plasma cells in TNBCs by assessing their association with IgG Kappa light chain expression, B cells, and TLS. METHODS:A retrospective analysis of 269 TNBC cases was performed. Tumour-infiltrating CD38+ plasma cells, CD20+ B cells, and TLS were evaluated on conventional haematoxylin-eosin-stained and immunohistochemical-stained sections of TNBC. We then selected TNBC cases demonstrating the highest and lowest densities of plasma cells, and examined their association with TLS, B cells, as well as immunoglobulin expression using Opal-Vectra multiplex immunofluorescence (IF). RESULTS:TNBC with high density of plasma cells showed significantly higher numbers of IgG Kappa+ CD38+ cells (p = 0.0089, p < 0.0001), and higher numbers of TLS (p < 0.0001), compared to TNBC with low density of plasma cells. TNBC with high density of plasma cells also showed higher numbers of CD20+ B cells in the tumour core (p < 0.0001), invasive margin (p < 0.0001), as well as stromal (p = 0.015) compartments. CONCLUSION:TNBC with high density of plasma cells are associated with higher numbers of IgG Kappa+ CD38+ cells, CD20+ B cells, and TLS. Further studies to characterize the function of plasma cell infiltrates and how they may interact with other tumour-infiltrating lymphocytes and TLS in TNBC may help improve existing immunotherapy strategies. 10.1007/s10549-020-05548-y
    Prognostic Significance of Tumor-Infiltrating Lymphocytes and the Tertiary Lymphoid Structures in HER2-Positive Breast Cancer Treated With Adjuvant Trastuzumab. Lee Hee Jin,Kim Joo Young,Park In Ah,Song In Hye,Yu Jong Han,Ahn Jin-Hee,Gong Gyungyub American journal of clinical pathology OBJECTIVES:Tumor-infiltrating lymphocytes (TILs) have prognostic significance in breast cancer. The tertiary lymphoid structure (TLS) is related to the influx of TILs, and expression of major histocompatibility complex (MHC) I in tumor cells is necessary for the effective action of TILs. METHODS:We retrospectively evaluated the relationship of TILs and TLS and the expression of MHC I in 447 HER2-positive breast cancers treated with chemotherapy and 1 year of trastuzumab. RESULTS:TILs were more abundant in hormone receptor (HR)-/HER2+ tumors than in HR+/HER2+ tumors. HR-/HER2+ breast cancers with abundant TILs showed a higher histologic grade, the absence of lymphovascular invasion, the presence of peritumoral lymphocytic infiltration, moderate to abundant TLSs in adjacent tissue, and stronger HLA-ABC and HLA-A expression. Abundant TILs and the absence of lymphovascular invasion were found to be good, independent prognostic factors for disease-free survival in patients with HR-/HER2+ breast cancer. The level of TILs was not associated with the patients' prognosis in HR+ tumors. CONCLUSIONS:Abundant TILs are an independent prognostic factor in HR-/HER2+ breast cancers. Evaluation of TILs in HR-/HER2+ breast cancers may provide valuable information regarding the prognosis of patients treated using adjuvant chemotherapy and trastuzumab. 10.1309/AJCPIXUYDVZ0RZ3G
    CD20CD22ADAM28 B Cells in Tertiary Lymphoid Structures Promote Immunotherapy Response. Frontiers in immunology Background:As the indication for immunotherapy is rapidly expanding, it is crucial to accurately identify patients who are likely to respond. Infiltration of B cells into many tumor types correlates with a good response to immune checkpoint inhibitor (ICI) therapy. However, B cells' roles in the anti-tumor response are far from clear. Methods:Based on single-cell transcriptomic data for ICI-treated patients, we identified a B-cell cluster [B (ICI-Responsive B) cells] and described the phenotype, cell-cell communication, biological processes, gene signature, and prognosis value of B cells through bioinformatic analysis, tissue immunofluorescence, and animal experiments. Surgery samples from 12 non-small cell lung carcinoma (NSCLC) patients with adjuvant checkpoint blockade were evaluated as external validation. Results:B cells were identified as a subset of CD20CD22ADAM28 B cells with a memory phenotype. Bioinformatic analysis revealed that B cells had enhanced cell viability and epigenetic regulation, and that ALOX5AP, MIF, and PTPRC/CD45 expressed by myeloid cells may be critical coordinators of diverse biological processes of B cells. Immunofluorescence confirmed the presence of B cells in tertiary lymphoid structures (TLSs) in skin SCC, RCC, CRC, and breast cancer. B-associated gene signatures correlate with positive outcomes in patients with melanoma, glioblastoma, NSCLC, HNSCC, or RCC treated with ICI therapy, and B-cell density predicted NSCLC patients' response to checkpoint immunotherapy. In line with this, melanoma-bearing mice depleted of B cells were resistant to ICIs. Conclusions:CD20CD22ADAM28 B cells were present in cancer-associated TLS and promoted the response to ICI therapy. 10.3389/fimmu.2022.865596
    The Presence of Tertiary Lymphoid Structures Provides New Insight Into the Clinicopathological Features and Prognosis of Patients With Breast Cancer. Frontiers in immunology Background:Tertiary lymphoid structures (TLSs) have been proven to be predictive biomarkers of favorable clinical outcomes and response to immunotherapies in several solid malignancies. Nevertheless, the effect of TLSs in patients with breast cancer (BC) remains controversial. The objective of the current study is to investigate the clinicopathological and prognostic significance of TLSs in BC. Given the unique difficulties for detecting and quantifying TLSs, a TLS-associated gene signature based on The Cancer Genome Atlas (TCGA) BC cohort was used to validate and supplement our results. Methods:Electronic platforms (PubMed, Web of Science, EMBASE, the Cochrane Library, CNKI, and Wanfang) were searched systematically to identify relevant studies as of January 11, 2022. We calculated combined odds ratios (ORs) with 95% confidence intervals (CIs) to determine the relationship between clinicopathological parameters and TLSs. The pooled hazard ratios (HRs) and 95% CIs were also calculated to evaluate the prognostic significance of TLSs. The TLS signature based on the TCGA BC cohort was applied to validate and supplement our results. Results:Fifteen studies with 3,898 patients were eligible for enrollment in our study. The combined analysis indicated that the presence of TLSs was related to improved disease-free survival (DFS) (HR = 0.61, 95% CI: 0.41-0.90, < 0.05) and overall survival (OS) (HR = 1.66, 95% CI: 1.26-2.20, < 0.001). Additionally, the presence of TLSs was positively correlated with early tumor TNM stage and high tumor-infiltrating lymphocytes. TLS presence was positively related to human epidermal growth factor receptor 2 (HER-2) and Ki-67 but inversely correlated with the status of estrogen and progesterone receptor. Simultaneously, our study found that tumor immune microenvironment was more favorable in the high-TLS signature group than in the low-TLS signature group. Consistently, BC patients in the high-TLS signature group exhibited better survival outcomes compared to those in the low-TLS signature group, suggesting that TLSs might be favorable prognostic biomarkers. Conclusions:TLS presence provides new insight into the clinicopathological features and prognosis of patients with BC, whereas the factors discussed limited the evidence quality of this study. We look forward to consistent methods to define and characterize TLSs, and more high-quality prospective clinical trials designed to validate the value of TLSs alone or in combination with other markers. 10.3389/fimmu.2022.868155
    Distinct Tertiary Lymphoid Structure Associations and Their Prognostic Relevance in HER2 Positive and Negative Breast Cancers. Liu Xia,Tsang Julia Y S,Hlaing Thazin,Hu Jintao,Ni Yun-Bi,Chan Siu Ki,Cheung Sai Yin,Tse Gary M The oncologist BACKGROUND:The presence of tumor infiltrating lymphocytes (TIL) is associated with favorable prognosis. Recent evidence suggested that not only their density, but also the spatial organization as tertiary lymphoid structures (TLS), play a key role in determining patient survival. MATERIALS AND METHODS:In a cohort of 248 breast cancers, the clinicopathologic association and prognostic role of TLS was examined. RESULTS:Tertiary lymphoid structures were associated with higher tumor grade, apocrine phenotype, necrosis, extensive in situ component, lymphovascular invasion (LVI), and high TIL. For biomarkers, TLS were associated with hormone receptors negativity, HER2 positivity, and c-kit expression. Tertiary lymphoid structures were significantly related to better disease-free survival (DFS) in HER2 positive (HER2+) breast cancers (log-rank = 4.054), which was not dependent on high TIL status. The combined TLS and TIL status was an independent favorable factor associated with DFS in those cases. Interestingly, tumor cell infiltration into the TLS was found in 41.9% of TLS positive cases. It was associated with LVI in HER2 negative (HER2-) TLS positive (particularly estrogen receptor positive [ER+] HER2-) cases. In the ER+ HER2- cases, tumor cell infiltration into TLS was also associated with increased pathologic nodal stage (pN) stage and nodal involvement. CONCLUSION:Tertiary lymphoid structures showed a similar relationship with clinicopathologic features and biomarkers as TIL. The presence of TLS, irrespective of TIL level, could be an important favorable prognostic indicator in HER2+ breast cancer patients. Given the significance of TLS in promoting effective antitumor immunity, further understanding of its organization and induction may provide new opportunities to improve the current immunotherapy strategies. IMPLICATIONS FOR PRACTICE:Despite recent interest on the clinical value of tumor infiltrating lymphocyte (TIL), little was known on the clinical significance on their spatial organization as tertiary lymphoid structures (TLS). Although TLS showed similar relationships with clinicopathologic features and biomarkers as TIL, the prognostic value of TLS, particularly in HER2 positive cancers, was independent of TIL. Moreover, tumor infiltration could be present in TLS which appears to be related to tumor invasion in HER2 negative cancers. Overall, the results demonstrated the additional value for TLS in HER2 cancer subtypes. Further investigations and its standardized evaluation will enhance its use as standard practice. 10.1634/theoncologist.2017-0029
    The prognostic significance of peritumoral tertiary lymphoid structures in breast cancer. Sofopoulos Michael,Fortis Sotirios P,Vaxevanis Christoforos K,Sotiriadou Nectaria N,Arnogiannaki Niki,Ardavanis Alexandros,Vlachodimitropoulos Dimitrios,Perez Sonia A,Baxevanis Constantin N Cancer immunology, immunotherapy : CII Tumors and their surrounding area represent spatially organized "ecosystems", where tumor cells and the immune contextures of the different compartments are in a dynamic interplay, with potential clinical impact. Here, we aimed to investigate the prognostic significance of peritumoral tertiary lymphoid structures (TLS) either alone or jointly with the intratumoral densities and spatial distribution of CD8 + and CD163 + cells in breast cancer (BCa) patients. TLS were identified peritumorally, within the area distancing up to 5 mm from the infiltrative tumor border, counted and further characterized as adjacent or distal, in formalin-fixed, paraffin-embedded tumor tissue samples from a cohort of 167 patients, with histologically confirmed invasive ductal BCa. TLS and tumor-infiltrating immune cells were determined by H&E and immunohistochemistry. Clinical follow-up was available for 112 of these patients. Patients with peritumoral TLS exhibited worse disease-free survival (DFS) and overall survival (OS) as compared to patients lacking TLS. Moreover, the density of peritumoral TLS was found to be crucial for prognosis, since patients with abundant TLS exhibited the worst DFS and OS. By combining the density of adjacent TLS (aTLS) with our recently published intratumoral signatures based on the differential distribution of CD8 + and CD163 + in the tumor center and invasive margin, we created two improved immune signatures with superior prognostic strength and higher patient population coverage. Our observations strengthen the notion for the fundamental role of the dynamic interplay between the immune cells within the tumor microenvironment (center/invasive margin) and the tumor surrounding area (peritumoral TLS) on the clinical outcome of BCa patients. 10.1007/s00262-019-02407-8
    Reliability of tumor-infiltrating lymphocyte and tertiary lymphoid structure assessment in human breast cancer. Buisseret Laurence,Desmedt Christine,Garaud Soizic,Fornili Marco,Wang Xiaoxiao,Van den Eyden Gert,de Wind Alexandre,Duquenne Sebastien,Boisson Anais,Naveaux Celine,Rothé Francoise,Rorive Sandrine,Decaestecker Christine,Larsimont Denis,Piccart-Gebhart Martine,Biganzoli Elia,Sotiriou Christos,Willard-Gallo Karen Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc The presence of tumor-infiltrating lymphocytes (TIL), reflecting host immune activity, is frequently correlated with better clinical outcomes, particularly in HER2-positive and triple-negative breast cancer. Recent findings suggest that organization of immune infiltrates in tertiary lymphoid structures also has a beneficial effect on survival. This study investigated inter- and intra-observer variation in TIL assessment using conventional hematoxylin-eosin versus immunohistochemical staining to identify immune cells. Global, intratumoral, and stromal TIL, as well as tertiary lymphoid structures were scored independently by experienced pathologists on full-face tumor sections (n=124). The fidelity of scoring infiltrates in core biopsies compared to surgical specimens, and pathological assessment compared to quantitative digital analysis was also evaluated. The inter-observer concordance correlation coefficient was 0.80 for global, 0.72 for intratumoral, and 0.71 for stromal TIL, while the intra-observer concordance correlation coefficient was 0.90 for global, 0.77 for intratumoral, and 0.89 for stromal TIL using immunohistochemical stains. Correlations were lower with hematoxylin-eosin stains, particularly for intratumoral TIL, while global scores had the highest concordance correlation coefficients. Our study concluded that tertiary lymphoid structures are accurately and consistently scored using immunohistochemical but not hematoxylin-eosin stains. A strong association was observed between TIL in core biopsies and surgical samples (R=0.74) but this did not extend to tertiary lymphoid structures (R=0.26). TIL scored by pathologists and digital analysis were correlated but our analysis reveals a constant bias between these methods. These data challenge current criteria for TIL and tertiary lymphoid structure assessment in breast cancer and recommend that how pathologists evaluate immune infiltrates be reexamined for future studies. 10.1038/modpathol.2017.43