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Immature B cells in the bone marrow emigrate into the spleen during adult lymphopoiesis. Here, we report that emigration is shifted to earlier B-cell stages in mice with orthotopic breast cancer, spontaneous ovarian cancer, and possibly in human breast carcinoma. Using mouse and human bone marrow aspirates and mouse models challenged with highly metastatic 4T1 breast cancer cells, we demonstrated that this was the result of secretion of thymic stromal lymphopoietin (TSLP) by cancer cells. First, TSLP downregulated surface expression of bone marrow (BM) retention receptors CXCR4 and VLA4 in B-cell precursors, increasing their motility and, presumably, emigration. Then, TSLP supported peripheral survival and proliferation of BM B-cell precursors such as pre-B–like cells. 4T1 cancer cells used the increased pool of circulating pre-B–like cells to generate metastasis-supporting regulatory B cells. As such, the loss of TSLP expression in cancer cells alone or TSLPR deficiency in B cells blocked both accumulation of pre-B–like cells in circulation and cancer metastasis, implying that the pre-B cell–TSLP axis can be an attractive therapeutic target.Significance:Cancer cells induce premature emigration of B-cell precursors from the bone marrow to generate regulatory B cells.

Neoadjuvant chemotherapy (NAC) is standard of care for patients with locally advanced breast cancer. TIL scoring is prognostic for response and has additional predictive value to the residual cancer burden after NAC. However, NAC induces various changes in the tumor bed and reliability of TIL scoring in post-NAC samples has not yet been studied. Pre- and post- NAC FFPE blocs from 60 patients were collected. H&E and dual CD3/CD20 chromogenic IHC-stained tissues were prepared and scored for global, stromal and intratumoral TIL by two experienced pathologists. Digital TIL scoring was preformed using the HALO® image analysis software. Comparison of the different scoring methods was done by calculation the concordance correlation coefficient (CCC), the Bland-Altman method and Passing-Bablok regression analysis. In patients with residual disease we demonstrate a good inter-pathologist correlation for global and stromal TIL on H&E stained tissue (CCC value 0.73, CI 0.53–0.85 for stromal TIL on H&E). Good correlation for scoring using the two staining methods (CCC 0.81, CI 0.67–0.90 for stromal TIL score) and with digital TIL scoring (CCC 0.77, CI 0.59–0.88) is demonstrated. There is only a poor to moderate reliability for intratumoral TIL scores. While overall concordance for TIL scoring in patients with a complete response is poor, there is a moderate inter-pathologist reliability for stromal TIL scoring on CD3/CD20 stained tissue. This study demonstrates a good reliability for TIL scoring in patients after NAC treatment, comparable to results in untreated breast cancer patients, in patients that have detectable residual tumor. Since there is a good concordance with digital TIL scoring, the development of a validated algorithm could be helpful in the future.

Background Ductal carcinoma in situ (DCIS) is considered a low-risk disease of the breast. Current increases in its incidence have resulted in many women either being under- or over-treated due to our limited findings on independent prognostic and predictive biomarkers. The Van Nuys Prognostic Index, based on tumor size, margin status, grade and age, is one tool used in treatment decisions. Patients with a low score show no significant benefit from radiotherapy, in contrast to those with an intermediate score, while patients with high scores should be considered for mastectomy. In contrast, for invasive ductal carcinoma (IDC) of the breast there is a strong consensus for the prognostic and predictive value of tumor infiltrating lymphocytes (TIL). As very little is known about TIL in DCIS, the goal of this study is to fully characterize the immune infiltrate and compare it to IDC, examine differences in the balance between effector and regulatory subpopulations and potentially discover new biomarkers for risk stratification. Material and Methods Fourteen patients were prospectively enrolled at the St. Luc hospital in Brussels, including 4 pure DCIS, 5 mixt DCIS and IDC, and 5 normal breast tissues. Formalin-fixed paraffin-embedded sections were stained with three fluorescent multiplex immunohistochemistry (mIHC) panels that combined antibodies to CD45, CD4, CD8, CD20, FOXP3, CD68, GZMB, PD-1, Ki67 and cytokeratin. InForm® Tissue Finder™ software and PhenoptrReports (Akoya Biosciences®) were employed for TIL quantification and spatial distribution. Freshly resected DCIS tissues were used to isolate tumor-infiltrating CD4 and CD8 T cells for single cell RNAseq analysis to determine the T cell clonotypes present (A. Devaux’s poster) Results Our analyses reveal the DCIS stroma has a significant immune infiltrate dominated by CD4+ helper T cells and B cells (140 and 115 cells/mm2, respectively) followed by CD8+ cytotoxic T cells (72 cells/mm2), regulatory T cells (Treg) (27 cells/mm2) and to a lesser extent macrophages (23 cells/mm2). The immune pattern in DCIS is similar to IDC except there are fewer macrophages in the tumor areas and Treg increase in the stroma. Tumor areas are generally less infiltrated than the stroma but some DCIS cells are in direct contact with T cells and macrophages. Spatial distribution analysis within a radius of 30 μm confirms that Treg are in close proximity to the DCIS cells and in the proximity of CD4+ helper and CD8+ cytotoxic T cells. Moreover, proliferating GZMB+ cells, mainly CD8+ cytotoxic T cells, were observed in direct contact with DCIS cells. Only one patient out of 4 had PD1+ TIL in the stroma. A comparison of pure and mixt DCIS reveals lower stromal infiltration by T and B cells in the former, which is also associated with an increase in macrophages. Finally, the abundance of stromal TIL was frequently organized in tertiary lymphoid structures (TLS), composed by a B cell follicle surrounded by a T cell zone containing both CD4+ helper and CD8+ cytotoxic T cells. TLS were characterized by the presence of proliferating B cells and PD1high T follicular helper cells. FOXP3+ and GZMB+ cells were also observed in the T cell zone. Conclusions Examination of the immune infiltrate in DCIS shows an abundance of helper T cells, B cells and active cytotoxic T cells in association with stromal TLS. These observations reveal an active tumor immune microenvironment in DCIS and suggest that the immune response plays an active role in DCIS pathogenesis. Citation Format: Soizic Garaud, Alix Devaux, Anais Boisson, Céline Naveaux, Pierre Coulie, Ahmad Awada, Karen Willard-Gallo. Characterization of the immune microenvironment in ductal carcinoma in situ of the breast [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-21.

Most breast cancers exhibit low immune infiltration and are unresponsive to immunotherapy. We hypothesized that inhibition of the receptor activator of nuclear factor-κB (RANK) signaling pathway may enhance immune activation. Here we report that loss of RANK signaling in mouse tumor cells increases leukocytes, lymphocytes, and CD8+ T cells, and reduces macrophage and neutrophil infiltration. CD8+ T cells mediate the attenuated tumor phenotype observed upon RANK loss, whereas neutrophils, supported by RANK-expressing tumor cells, induce immunosuppression. RANKL inhibition increases the anti-tumor effect of immunotherapies in breast cancer through a tumor cell mediated effect. Comparably, pre-operative single-agent denosumab in premenopausal early-stage breast cancer patients from the Phase-II D-BEYOND clinical trial (NCT01864798) is well tolerated, inhibits RANK pathway and increases tumor infiltrating lymphocytes and CD8+ T cells. Higher RANK signaling activation in tumors and serum RANKL levels at baseline predict these immune-modulatory effects. No changes in tumor cell proliferation (primary endpoint) or other secondary endpoints are observed. Overall, our preclinical and clinical findings reveal that tumor cells exploit RANK pathway as a mechanism to evade immune surveillance and support the use of RANK pathway inhibitors to prime luminal breast cancer for immunotherapy., info:eu-repo/semantics/published

The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) is now widely accepted and being implemented in clinical practice. Our lab previously demonstrated that 60% of BC organize some of their TIL in tertiary lymphoid structures (TLS). TLS have been detected in a wide variety of solid tumors with their prognostic value and importance in the response to immunotherapy increasingly accepted. Reliable biomarkers to identify and characterize TLS and their immune activities in tumors are needed. The specific aim of this project was to perform a comprehensive analysis across the spectrum of TLS states to identify biomarkers associated with active and inactive TLS, where the former are associated with functional anti-tumor immunity and improved responses to treatment and long-term survival. This study analyzed FFPE tissues from 38 primary untreated HER2+ and triple negative (TN) BC patients. Patients were divided into two groups based on a TLS score evaluating CD3/CD20 and PD-1/Ki-67 dual IHC-stained tumor tissues: 1) BC containing a majority of active TLS (tumors with only active TLS have never been observed), defined by the presence of a germinal center (GCpos ; Ki-67+ follicular B cells) and 2) BC with only inactive TLS (GCneg ; Ki-67− follicular B cells). Controls included normal breast tissues, BC without TLS and lymphoid tissues (reactive tonsils for GCpos and spleens for GCneg TLS). RNA extracts from microdissected tissues (N=100), including active TLS, inactive TLS and lymphoid B follicles, were sequenced. DESeq2 and CIBERSORT were employed to quantify differentially expressed genes and immune cell subpopulations, respectively. Gene Set Enrichment Analysis was used for additional data interpretation and pathway identification. Tumors from patients with active TLS were characterized by increased naïve and plasma B cell TIL compared to tumors with only inactive TLS. Preliminary analysis of the differentially expressed genes in active TLS (vs inactive TLS) revealed upregulation of key B cell differentiation, somatic hypermutation and class switch recombination genes, which paralleled their respective lymphoid controls. These gene upregulations were linked with a GC presence in active TLS. A specific set of immunoglobulin genes were also differentially expressed in active TLS. Continued analysis of the TLS data, including their cellular composition, location, maturation and functionality, along with data confirmation (RT-PCR and multiplex IHC), and assessment of their functional and clinical relevance is ongoing. Our preliminary results suggest that active B cell differentiation and Ig production contribute to TLS functionality. In lymphoid tissues, the GC plays important roles in orchestrating the molecular and cellular programs of humoral immunity. Our data suggest that active GC-containing TLS foster an immune microenvironment in BC that favors positive clinical outcomes. Citation Format: Doïna Sofronii, Francine Padonou, Mireille Langouo, Noemie Thomas, Anais Boisson, Alexandre De Wind, Denis Larsimont, Ahmad Awada, Soizic Garaud, Karen Willard-Gallo. Biomarkers of functionally active tertiary lymphoid structures in human breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4618.

Background: Optimal dosing and duration of adjuvant treatment with PD-1 and CTLA-4 immune checkpoint inhibitors have not been established. Prior to their regulatory approval we investigated a low-dose regimen of nivolumab with or without ipilimumab in a sequential dual-cohort phase II clinical trial. Methods: Following the complete resection of melanoma metastases, patients were treated with a single fixed dose of ipilimumab (50 mg) plus 4 bi-weekly fixed doses of nivolumab (10 mg) (cohort-1), or nivolumab for 1 year (10 mg fixed dose, Q2w x9, followed by Q8w x4) (cohort-2). Twelve-months relapse-free survival (RFS) served as the primary endpoint. Results: After a median follow-up of 235 weeks for cohort-1 (34 patients), and 190 weeks for cohort-2 (21 patients), the 12-months RFS-rate was, respectively, 55.9% (95% CI, 39–72), and 85.7% (95% CI, 70–100). Treatment-related adverse events occurred in 27 (79%), and 18 (86%) patients, with 3 (9%), and 1 (5%) grade 3 adverse events in cohort-1 and -2, respectively. Immunohistochemical quantification of intra- and peritumoral CD3+ T cells and CD20+ B cells, but not PD-1/PD-L1 staining, correlated significantly with RFS. Conclusions: One year of adjuvant low-dose nivolumab could be an effective and economically advantageous alternative for standard dosing, at the condition of further confirmation in a larger patient cohort. A shorter low-dose nivolumab plus ipilimumab regimen seems inferior and less tolerable.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4(+) Tfh TIL, CD8(+) TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1(hi)ICOS(int) phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67(+) TIL-B in active germinal centers) and cytotoxic (GZMB(+)CD8(+) and GZMB(+)CD68(+) TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25(+)CXCR5(+)GARP(+)FOXP3(+) phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β–dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8(+) TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N-6-methyladenosine (m(6)A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m(6)A and altered 3-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors.Fuks and colleagues report that downregulation of the FTO m(6)A RNA demethylase activates the Wnt pathway, promoting EMT-mediated progression of epithelial tumors and conferring sensitivity to Wnt inhibitors.

Using the immune system to fight cancer has garnered tangible success, but some treatments, like neoadjuvant chemotherapy (NAC), modulate the immune microenvironment. Recent studies show that the spatial organization of tumor infiltrating lymphocytes (TIL) have greater predictive value than TIL density. The effect of NAC on immune composition and spatial distribution is not fully understood but new insight could help to guide its use in combination with immune therapy and identify patients with potential to derive benefit. We spatially profiled 84 RNA targets (GeoMx®) in a cohort of 12 NAC-treated breast cancer patients (4 Luminal, 4 HER2+ and 4 triple negative), none of whom achieved a pathological complete response. Matched pre- and post-treatment tissue samples were analyzed together with regions of interest (tumor center, invasive margin and TIL aggregates) identified using CD3, CD20, Syto83 and pan-cytokeratin for stromal/tumor segmentation. NAC decreases overall gene expression in breast tumors with the biggest declines seen in tumor promoting (CCND1, AKT1, CTNNB1, EPCAM, VEGFA, KRT and MKI67) and some inflammatory (CXCL10, STAT1 and STAT2) genes (p-value 50% cellularity). Poor responders expressed higher levels of tumor promoting genes pre-NAC, which remained high after treatment (KRT p=0.023, CTNNB1 p=0.031). No differences were detected in immune genes in the stroma based on patient responsiveness; however, higher antigen presentation and inflammatory gene transcripts were found at the tumor margins of good responders. Post-NAC differences between the margin and center decrease in good responders paralleled by a shift towards higher or equal expression of some inflammatory markers at the tumor center. Poor responders maintain high expression of all immune markers at the margin. A higher number of aggregates (mean n=5 vs n=1.3) were detected in good compared to poor responders together with more tertiary lymphoid structures (mean n=2.4 vs n=0.3) and distinguished by higher immune gene expression (CD8 p=0.046, CCL5 p=0.054, NKG7 p=0.022). NAC induces changes in other cells in the tumor microenvironment while targeting tumor cells. Our data show that spatial analysis of gene expression comparing good and poor responders (without a pathological complete response) reveal that tumor cells in the latter retain expression of tumor promoting genes while the immune compartment remains excluded. Good responders are characterized by a decrease in tumor promoting genes in parallel with lymphoid aggregates, including TLS, of active immune cells in the stroma and at the tumor center. These findings suggest that tailoring adjuvant treatment between good and poor responding patients might be warranted. Citation Format: Noémie Thomas, Soizic Garaud, Mireille Langouo, Ioannis Zerdes, Doïna Sofronii, Anaïs Boisson, Theodoros Foukakis, Alexandre De Wind, Roberto Salgado, Ahmad Awada, Karen Willard-Gallo. Spatial organization of the immune microenvironment after neoadjuvant treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2045.

Introduction While immune infiltrates have already been extensively characterized in primary tumors (P), data on breast cancer metastases (M) remains limited. To this end we quantified and qualified the immune cells in a unique cohort of multiple matched P and M samples selected from an institutional breast cancer autopsy cohort. Patients and methods Twenty-three patients were selected from an institutional autopsy program (Semmelweis University, Budapest, Hungary) based on matched P and M sample availability (124 samples). All samples were centrally characterized for estrogen (ER), progesterone (PR) and HER2 receptors. The primary molecular subtypes were as follows: 9 ER+/PR+/HER2-, 8 triple negative and 6 HER2+. Ten patients relapsed ≤1 year after diagnosis and were further referred to as “early relapsers”, as opposed to the remaining qualified as “late relapsers”. Immunohistochemistry (IHC) was carried out against CD3/CD20 and CD4/CD8 in 21 patients (119 samples). Tumor infiltrating lymphocytes (TILs) were assessed on hematoxylin and eosin (H&E) and CD3-stained slides. Gene expression data were generated using the NanoString nCounter assay (PanCancer Immune Profiling Panel) for 11 patients (35 samples) and analyzedusing the R package NanoStringQCPro. The scores from published immune gene signatures were calculated as a weighted sum of the expressions of their genes. All samples were analyzed for 22 immune cell subtypes relative abundance using CIBERSORT. Results TILs assessed on H&E and CD3-stained slides were weakly correlated (Rho= 0.38, p Conclusion This is to the best of our knowledge the first study characterizing the immune infiltration in patients with multiple matched P and M samples. The results suggest that Ms have not only a globally lower immune infiltration as compared to Ps, but also a different immune composition. Additionally, Ms from late relapsers are more infiltrated as compared to early relapsers. The present data also uncovers not only important inter-patient but also intra-patient immune heterogeneity, which should be taken into consideration for optimal treatment decision. Citation Format: Szekely B, Bareche Y, Van den Eynden G, Salgado R, Buisseret L, Garaud S, Willard-Gallo K, Hatzis C, Szasz M, Kulka J, Larsimont D, Sotiriou C, Pusztai L, Desmedt C. Immune characterization of matched primary and multiple metastatic samples issued from an institutional autopsy cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-10.

The clinical utility of tumor-infiltrating lymphocytes (TIL) is actively being investigated in breast cancer (BC). It is unclear whether TIL spatial location and organization in tertiary lymphoid structures (TLS) have an impact on prognosis. Additionally, the significance of PD-1 and PD-L1 expression is being debated due to conflicting data from several studies. We hypothesize that the presence, extent and spatial location of multiple immune biomarkers, reflecting ongoing immune responses, will be consistently associated with a good prognosis in highly infiltrated BC [triple-negative (TNBC) and HER2+]. The relationship between these immune biomarkers and clinical outcome was examined in the TNBC and HER2+ cohorts of node-positive BC patients enrolled in the BIG 02-98 adjuvant phase III trial with available material for immunohistochemical (IHC) labeling (N=113 and N=136, respectively). HER2+ patients did not receive trastuzumab. Dual IHC staining was performed on full-face consecutive tissue sections. Scoring was independently performed by two pathologists, blinded to the clinical data, and included: global, intratumoral and stromal TIL and TLS, assessed on CD3/CD20 slides; the percentage and location of PD-1 and PD-L1 expression, assessed on PD-1/PD-L1 slides. TIL were considered as a categorical variable with different cut-offs used for each parameter and for each cohort (TNBC and HER2+). Invasive disease-free survival (I-DFS) and overall survival (OS) were analyzed (median follow-up: 10 years). Cox proportional hazard models were used for survival analyses. The TNBC cohort revealed an association between global TIL and outcome [adjusted hazard ratio (HR) for I-DFS: 0.27 (0.15-0.51); OS: 0.26 (0.13-0.53)]. Similar results were observed for stromal and intratumoral TIL. PD-L1 expression within TLS was an independent predictor of OS, after adjustment for tumor size and age [HR: 0.30 (0.09-0.99)]. Multivariate analysis reveals this effect was principally driven by high stromal TIL (>17.5% based on CD3/CD20 assessment) (χ2 OS: p=0.009). In contrast, no significant prognostic associations were found in the overall HER2+ cohort. However high T cell TIL were associated with improved I-DFS and OS in the ER-/HER2+ group [I-DFS: 0.34 (0.14-0.80); OS: 0.32 (0.12-0.86)] and stromal TIL were associated with improved I-DFS in the ER+/HER2+ group [HR: 0.29 (0.09-0.94)] (univariate analyses). No significant associations between the number of TLS nor the expression of PD-1 with outcomes were observed in either cohorts. The presence of PD-L1+ TLS, driven by high baseline TIL, was associated with an excellent prognosis in node-positive TNBC. This observation might reflect specific immune activities taking place in these mini lymph node-like structures adjacent to the tumor bed where specific antitumor memory immune responses could be generated. No different prognostic impact was observed when analyzing TIL spatial location. Although the statistical power of the study might be limited, in line with previous findings our data reveal that, among the immune parameters evaluated, TIL are the strongest predictor of outcome in TNBC, while PD-L1+ TLS could be a new and important parameter that requires further investigation. Citation Format: Solinas C, de Wind A, Van den Eynden G, Ameye L, Garaud S, De Silva P, Boisson A, Noel G, Langouo Fontsa M, Buisseret L, de Azambuja E, Francis PA, Di Leo A, Crown JP, Sotiriou C, Larsimont D, Paesmans M, Piccart-Gebhart M, Willard-Gallo K. Immune parameters associated with survival in triple negative and HER2-positive breast cancer patients with 10 years of follow-up [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-09.

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

Tumor-infiltrating B-cells (TIL-B) in breast cancer (BC) have previously been associated with improved clinical outcomes; however, their role(s) in tumor immunity is not currently well known. This study confirms and extends the correlation between higher TIL-B densities and positive outcomes through an analysis of HER2-positive and triple-negative BC patients from the BIG 02-98 clinical trial (10yr mean follow-up). Fresh tissue analyses identify an increase in TIL-B density in untreated primary BC compared to normal breast tissues, which is associated with global, CD4+ and CD8+ TIL, higher tumor grades, higher proliferation and hormone receptor negativity. All B-cell differentiation stages are detectable but significant increases in memory TIL-B are consistently present. BC with higher infiltrates are specifically characterized by germinal center TIL-B, which in turn are correlated with TFH TIL and antibody-secreting TIL-B principally located in tertiary lymphoid structures. Some TIL-B also interact directly with tumor cells. Functional analyses reveal TIL-B are responsive to BCR stimulation ex vivo, express activation markers and produce cytokines and immunoglobulins despite reduced expression of the antigen-presenting molecules HLA-DR and CD40. Overall, these data support the concept that ongoing humoral immune responses are generated by TIL-B and help to generate effective anti-tumor immunity at the tumor site. ispartof: JCI INSIGHT vol:4 issue:18 ispartof: location:United States status: published

Background: Immunotherapy in the form of immune checkpoint inhibition (ICI) is now well established as acornerstone for treating many advanced malignancies. Nevertheless, as the number of indications for checkpoint inhibitors increases, so does the risk of immune-related adverse events (irAEs). Methods: We report two patient cases who, after being treated by an anti-programmed cell death 1 (PD-1), presented with grade III dyspnea due to pneumonitis. Discussion: Immunotherapy was discontinued and the patients required treatment with systemic corticosteroids. At the time of writing, both patients are still in complete response (CR), more than 1year beyond immunotherapy discontinuation. We discuss our cases with regard to recent literature reports on immune-related pneumonitis and persistence of response beyond discontinuation.

Immature B cells in the bone marrow emigrate into the spleen during adult lymphopoiesis. Here, we report that emigration is shifted to earlier B-cell stages in mice with orthotopic breast cancer, spontaneous ovarian cancer, and possibly in human breast carcinoma. Using mouse and human bone marrow aspirates and mouse models challenged with highly metastatic 4T1 breast cancer cells, we demonstrated that this was the result of secretion of thymic stromal lymphopoietin (TSLP) by cancer cells. First, TSLP downregulated surface expression of bone marrow (BM) retention receptors CXCR4 and VLA4 in B-cell precursors, increasing their motility and, presumably, emigration. Then, TSLP supported peripheral survival and proliferation of BM B-cell precursors such as pre-B–like cells. 4T1 cancer cells used the increased pool of circulating pre-B–like cells to generate metastasis-supporting regulatory B cells. As such, the loss of TSLP expression in cancer cells alone or TSLPR deficiency in B cells blocked both accumulation of pre-B–like cells in circulation and cancer metastasis, implying that the pre-B cell–TSLP axis can be an attractive therapeutic target. Significance: Cancer cells induce premature emigration of B-cell precursors from the bone marrow to generate regulatory B cells.

There is a growing interest in active immune responses generated by tertiary lymphoid structures (TLS) arising in solid tumors; however, their clinical impact in breast cancer (BC) remains unclear. Several studies show that transcription factors contribute to TLS formation via their regulation of cytokine and chemokine production. The Forkhead box (FOX) protein 1 (FOXP1) has been shown to play critical roles in regulating immune cells, including our recent work revealing its effects on TIL migration. These data lead us to further investigate FOXP1 expression in tumor infiltrating lymphocytes (TIL) and TLS. We identify two types of TLS based on FOXP1 expression: 1) those that contain a germinal center (GC+) and those that do not (GC-). Comparative analysis of FOXP1 expression in secondary lymphoid organs, including more immune active tonsils (many GC) and less immune active spleens (primarily without GC) confirm differences in FOXP1 expression associated with GC. In BC, TLS containing tumors were more frequently GC- than GC+ (n=49), with triple-negative tumors having higher numbers of GC+ TLS compared to luminal or HER2+ tumors. Immunofluorescence and multiplex immunohistochemistry was used to closely examine the GC+ and GC- TLS, finding an immune active profile in the former, characterized by T follicular helper cells (PD1+CD4+ T), mature dendritic cells (CD21+ and CD23+), actively proliferating (Ki67+) B cells undergoing immunoglobulin (Ig) class switch recombination (AID+) and a plasma cell presence (CD138+). Analysis of Ig's in the primary tumor supernatants revealed that BC with ≥1 GC+ TLS (n=20) were characterized by increases in total Ig, IgG1, IgG2 and IgA, reflecting active humoral immunity, compared to BC containing only GC- TLS (n=29). Gene expression analysis of individual micro-dissected TLS demonstrated upregulation of Th1, Th2 and Tfh immune genes in the GC+ compared to the GC- TLS, suggesting the former also sustain cell-mediated immune responses. Immune infiltrates in tumors with ≥1 GC+ TLS are specifically characterized by high global TIL, CD3+, CD4+ or CD8+ T cell TIL and CD20+ TIL-B (n=29). Analysis of BC TIL spatial distribution identified increased stromal TIL (all subpopulations) while intratumoral TIL increases were predominantly CD3+ and CD8+ T cell TIL in tumors with GC+ TLS. Overall, our data indicate that GC+ TLS house active immune responses in BC while GC- TLS are quiescent. Citation Format: Pushpamali De Silva, Soizic Garaud, Cinzia Solinas, Grégory Noël, Mireille Langouo Fontsa, Anaïs Boisson, Alexandre de Wind, Vinu Jose, Gert Van den Eynden, Noemie Thomas, Hugues Duvillier, Céline Naveaux, Ligia Craciun, Dominique Bron, Martine Piccart-Gebhart, Denis Larsimont, Karen Willard-Gallo. The anti-tumor immune responses by active and quiescent tertiary lymphoid structures to breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3853.

We previously reported that breast cancer (BC) generates metastasis-promoting regulatory B cells (CD25+ tumor-evoked Bregs). Because phenotypically these cells resembled bone marrow (BM) Rag+ B-cell precursors, such as large Pre-B cells, we hypothesized whether cancer uses them to generate Bregs. To investigate this idea, we used mouse and human BM aspirates, human PBMCs from BC patients, and mice with different orthotopic cancers, such as highly metastatic 4T1 breast cancer cells and EMT6. We report that breast cancer indeed uses BM B-cell precursors to generate Bregs. However, to do this, cancer expresses thymic stromal lymphopoietin (TSLP). Mechanistically, cancer-produced TSLP downregulates surface expression of CXCR4 and α4β1 (VLA-4) of B-cell precursors, thereby impairing their BM retention and increasing their exit into the circulation. TSLP also enables survival and proliferation of these BM emigrants, while other factors such as 5-lipoxygenase metabolites convert them into Bregs. The loss of TSLP expression in cancer cells alone or TSLPR deficiency in B cells not only reduce pre-B cells in the circulation, but also retard lung metastasis. Overall, our data reveal a previously unknown function of cancer-produced TSLP as inducers of premature B-cell precursor emigration from BM to support Breg generation. The results also suggest that the TSLP-pre-B cell axis can be an attractive therapeutic target. Citation Format: Emeline Ragonnaud, Kanako Moritoh, Monica Bodogai, Soizic Garaud, Chen Chen, Xin Wang, Karen Willard-Gallo, Arya Biragyn. Cancer targets early B-cell precursors to generate metastasis-promoting Bregs by promoting their premature emigration from the bone marrow and expansion in the circulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4499.

An important role for tumor infiltrating B lymphocytes (TIL-B) in the immune response to cancer is emerging; however, very little is known about the antigen specificity of antibodies produced in situ. The presence of IgA antibodies in the tumor microenvironment has been noted although their biological functions and clinical significance are unknown. This study used a 91-antigen microarray to examine the IgG and IgA autoantibody repertoires in breast cancer (BC). Tumor and adjacent breast tissue supernatants and plasma from BC patients together with normal breast tissue supernatants and plasma from healthy controls (patients undergoing mammary reduction and healthy blood donors) were analyzed to investigate relationships between autoantibodies and the clinical, histological and immunological features of tumors. Our data show that >84% of the BC samples tested contain autoantibodies to one or more antigens on the array, with ANKRD30BL, COPS4, and CTAG1B being most frequently reactive. Ex vivo TIL-B responses were uncoupled from systemic humoral responses in the majority of cases. A comparison of autoantibody frequencies in supernatants and plasma from patients and controls identified eight antigens that elicit BC-associated autoantibody responses. The overall prevalence of IgG and IgA autoantibodies was similar and while IgG and IgA responses were not linked they did correlate with distinct clinical, pathological and immunological features. Higher levels of ex vivo IgG responses to BC-associated antigens were associated with shorter recurrence-free survival (RFS), HER2 overexpression and lower tumor-infiltrating CD8+ T cell counts. Higher IgA levels were associated with estrogen and progesterone receptor-negative cancers but were not significantly associated with RFS. Furthermore, ex vivo IgA but not IgG autoantibodies reactive to BC-associated antigens were linked with germinal center and early memory B cell maturation and the presence of tertiary lymphoid structures suggesting that these TIL-B are activated in the tumor microenvironment. Overall, our results extend the current understanding of the antigen specificity, the biological and the clinical significance of IgG and IgA autoantibodies produced by BC TIL-B in situ., SCOPUS: ar.j, info:eu-repo/semantics/published

Cancer immunotherapy is demonstrating impressive clinical benefit in different malignancies and clinical oncologists are increasingly turning their attention to immune-oncology. It is now well recognized that innate and adaptive immune cells infiltrating tumors are associated with clinical outcomes and responses to treatments, and can be harnessed to patients' benefit. Considerable advances have also been made in understanding how cancers escape from immune attack. Targeting of immunological escape processes regulated by the expression of immune checkpoint receptors and ligands and the down-modulation of tumor antigen presentation is the basis of immuno-oncology treatments. Despite recent achievements, there remain a number of unresolved issues in order to successfully implement cancer immunotherapy in many cancers. Importantly, clinical biomarkers are still needed for better optimization of emerging combination immunotherapies and better treatment tailoring. In this review, we summarize the function of innate and adaptive immune cells in anti-tumor immunity and the general mechanisms exploited by tumor cells to escape and inhibit immune responses as well as therapeutic strategies developed to overcome these mechanisms and discuss emerging biomarkers in immuno-oncology.

Background Recent work has shown that immune activity in breast cancer (BC) plays a role in treatment responses and long-term outcomes. TIL scoring is now implemented as a prognostic marker in clinical practice. Studies show that high tumor infiltrating lymphocytes (TIL) are more often detected in triple negative and HER2 positive subtypes. TIL contain a heterogeneous population of immune cells dominated by T cells but also consist of many others. Neoadjuvant therapy (NAT) is increasingly administered in patients with locally advanced BC, allowing a resection of the tumor and to evaluate the in vivo response. While cytotoxic agents such as Anthracyclines elicit immunogenic cell death, the impact of NAT on TIL has only been sporadically studied. Methods A detailed assessment of the tumor microenvironment (TME) includes evaluation of TIL density, composition and functionally. Following NAT, lymphocyte subpopulations were quantified using flow cytometry(FACS) and these data were compared to untreated BC patients. Tumor tissue supernatants were stored for assessment of immunoglobulins (Ig). The FFPE blocks from matched biopsies and surgical tissues were stained with HE, chromogenic IHC and multiplex IHC. The stromal and intratumoral TIL were scored by two experienced pathologists. Results Our preliminary data correlate better responses with highly infiltrated biopsies, consistent with published work. Overall, the trend following NAT is for a decrease in TIL density, but with great inter-patient variation. FACS analysis of immunophenotypes shows a shift from CD4 to CD8 T cells and an increase in memory B cells after NAT. An assay for Ig subtypes detected decreases in IgG1/IgM and increases in IgG2/IgA. Multiplex IHC analysis of the TME in a patient with residual cancer detected active immune responses surrounding the remaining tumor that were characterized by CD8 TIL attached to the malignant cells, suggesting their recognition and killing. Conclusion NAT provokes changes in immune activities at the tumor site in early BC, with some of these alterations apparently linked to specific treatments. Our ongoing efforts are designed to provide detailed insight into the alterations associated with different NAT modalities and define prognostic biomarkers. Legal entity responsible for the study The authors. Funding FRNS, Televie, Les Amis de l'Institut Bordet, Lambeau Marteau. Disclosure A. Pabois: Full / Part-time employment: Iteos therapeutics. All other authors have declared no conflicts of interest.

The remarkable responses observed in metastatic cancer patients treated with immunotherapies, including inhibitors directed to the PD-1 and PD-L1 checkpoint molecules, makes it a priority to identify critical variations in pro- and anti-tumor immune responses in breast cancer (BC). In patients with triple negative (TN) BC, an increased presence of tumor infiltrating lymphocytes (TIL) and tertiary lymphoid structures (TLS) have been associated with good clinical outcomes. However, the frequency of specific lymphocyte subpopulations, PD-1 and/or PD-L1 expression and their prognostic significance remains an open question. Our recent work found that PD-1 and PD-L1 expression are specifically associated with higher TIL densities and an increased number of TLS in BC. We further demonstrated that TIL density, TLS and PD-L1 expression were correlated with more aggressive breast tumor characteristics, including higher proliferation and hormone receptor negativity. In this project, we examined the prevalence of TIL, TLS, PD-1 and PD-L1 expression in TNBC and further compared these immune parameters between TNBC patients harboring BRCA1 or BRCA2 germline gene mutations with those carrying the wild-type (wt) genes. A total of 1402 BC patients whose blood was genetically tested for germline BRCA1 and BRCA2 mutations were examined for inclusion in this study. Ninety-eight chemotherapy-naïve patients with primary invasive ER–, PR– and HER2– BC and demonstrated germline BRCA1 or BRCA2 wt or mutated-gene status were included in this study. Ninety-four tumors were determined to be suitable for evaluating immune cell infiltration (51 BRCA wt and 43 BRCA-mutated). FFPE tumor tissue from the surgical specimens was analyzed by immunohistochemistry (IHC) staining of full-face tissue sections. IHC was performed as a dual label using CD3 plus CD20 for T and B cells, CD4 plus CD8 for the major T cell subpopulations and PD-1 plus PD-L1 for individual or paired expression of these receptors. The stained slides were independently scored by two experienced pathologists for TIL, TIL subpopulations, TLS and checkpoint molecule expression. These analyses revealed that 87% of our TNBC cohort was TIL-positive (≥10% TIL) with 35% classified as lymphocyte predominant BC (LPBC; ≥50% TIL). T cells were the principal component of the lymphocytic infiltrate with no significant differences between the BRCA wt and BRCA-mutated groups detected in total T cells (CD3+), helper T cells (CD4+), cytotoxic T cells (CD8+) or B cells (CD20+). TLS were identified in 73% of tumors with again no significant differences between the BRCA groups. Examination of checkpoint molecule expression identified 33% tumors as PD-1 positive and 40% as PD-L1 positive. PD-1 expression was correlated with PD-L1 expression and both with TIL positivity and the level of immune infiltration but not BRCA mutational status. Overall, our analyses revealed that BRCA wt and BRCA-mutated TNBC are remarkably similar in terms of TIL heterogeneity, a TLS presence and checkpoint molecule expression. These data suggest that BRCA gene mutations are not immunogenic nor do they directly drive immune infiltration in TNBC. Citation Format: Willard-Gallo K, Solinas C, Marcoux D, t'Kint de Roodenbeke D, Garaud S, Van den Eynden G, de Wind A, Boisson A, Larsimont D, Piccart M. BRCA gene mutations do not shape the extent and organization of tumor infiltrating lymphocytes in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-04.

Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer after invasive ductal cancer (IDC). Here, we aimed at evaluating the prevalence, levels, and composition of tumor-infiltrating lymphocytes (TILs) and their association with clinico-pathological and outcome variables in ILC, and to compare them with IDC., SCOPUS: ar.j, info:eu-repo/semantics/published

BACKGROUND: CD73 is an ecto-enzyme that promotes tumor immune escape through the production of immunosuppressive extracellular adenosine in the tumor microenvironment. Several CD73 inhibitors and adenosine receptor antagonists are being evaluated in phase I clinical trials. PATIENTS AND METHODS: Full-face sections from formalin-fixed paraffin-embedded primary breast tumors from 122 samples of triple-negative breast cancer (TNBC) from the BIG 02-98 adjuvant phase III clinical trial were included in our analysis. Using multiplex immunofluorescence and image analysis, we assessed CD73 protein expression on tumor cells, tumor-infiltrating leukocytes and stromal cells. We investigated the associations between CD73 protein expression with disease-free survival (DFS), overall survival (OS) and the extent of tumor immune infiltration. RESULTS: Our results demonstrated that high levels of CD73 expression on epithelial tumor cells were significantly associated with reduced DFS, OS and negatively correlated with tumor immune infiltration (Spearman’s R= −0.50, P

Ten-eleven translocation enzymes (TET1, TET2, and TET3), which induce DNA demethylation and gene regulation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are often down-regulated in cancer. We uncover, in basal-like breast cancer (BLBC), genome-wide 5hmC changes related to

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.

Background Breast cancer (BC) in young women has unique biology and poor prognosis. Previous reports suggest that they often express RANKL, which was also shown to play a role in mammary tumorigenesis and various immune processes. Here, we present the primary results of D-BEYOND, a window study investigating the biological activity of the RANKL inhibitor; denosumab in pre-menopausal BC patients. Methods D-BEYOND is a prospective, phase Iia, single-arm, multicenter study assessing the effect of denosumab on BC biology in premenopausal women with early BC (NCT01864798). Patients received two subcutaneous injections of denosumab (120mg), one week apart, followed by breast surgery. The primary endpoint was geometric mean change in tumor Ki67 assessed by immunohistochemistry (IHC). Blood, tumor and normal adjacent breast tissue were collected pre- and post-treatment. Serum levels of RANKL, OPG and CTX were assessed by ELISA. RNA was extracted from fresh-frozen tissue and RNAseq was performed. DESeq2 was used for differential expression analysis, GAGE was used for pathway analysis and CIBERSORT was used to infer immune cell subsets between pre- and post-treatment. Ki67, CD4/Foxp3 and CD4/CD8 IHC were performed on FFPE tissue to further assess the immune microenvironment. The percentage of TILs was independently evaluated by two pathologists on H&E slides. Pre- and post-treatment values were compared using a paired t-test. Results A total of 27 patients were enrolled in the study between October 2013 and July 2016. The median age was 45 years (range 35-51 years). Tumors of 21 patients were hormone receptor positive (77.8%), 4 were HER2 positive (14.8%) and 2 were triple negative (7.4%). No serious adverse events were reported, the most frequent non-serious adverse event being arthralgia (14.8%). After treatment, serum levels of CTX and RANKL decreased in all patients (P < 0.001) whereas OPG increased in 76.9% of patients (P = 0.009, 95% CI 0.56-0.91). There was no significant reduction of Ki67 values from baseline (geometric mean [GM] change after treatment; 0.98, 95% CI 0.76-1.26; P = 0.90). Twenty-four pre- and post-treatment tumor pairs were available for RNAseq, IHC and TILs evaluation. There was a significant increase in the percentage of stromal TILs after treatment (GM change of 1.75, 95% CI 1.28–2.39; P = 0.001). 1084 differentially expressed genes were identified and pathway analysis revealed enrichment of several immune processes. CIBERSORT revealed an enrichment of CD8+ T cells (GM change 1.72, 95% CI 1.19–2.48; P = 0.006) and a decrease of Treg cells (0.71, 95% CI 0.52–0.98, P = 0.040). These results were confirmed by IHC of CD8+ and CD4+/Foxp3+ cells (GM change 1.59, 95% CI 1.14–2.21; P = 0.008 and 0.63, 95% CI 0.49–0.83, P = 0.001, respectively). Conclusion Short course of denosumab did not reduce tumor proliferation rate. However, it induced a significant increase in TILs and CD8 cytotoxic T cells, while Treg infiltration decreased. These findings suggest an immunomodulatory role for denosumab in young breast cancer and that its use in combination could boost immunotherapy efficacy. Citation Format: Nguyen B, Marion M, Salgado R, Venet D, Vuylsteke P, Polastro L, Wieldiers H, Simon P, Lindeman G, Larsimont D, Van den Eynden G, Velghe C, Rothe F, Garaud S, Michiels S, Willard-Gallo K, Azim Jr HA, Loi S, Piccart M, Sotiriou C. The immunomodulatory potential of denosumab in breast cancer: results from D-BEYOND, a window of opportunity trial evaluating a RANK-ligand (RANKL) inhibitor and its biological effects in young pre-menopausal women diagnosed with early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-06.

Background: The association between tumor infiltrating lymphocytes (TIL) and positive clinical outcomes in breast cancer (BC) is now commonly recognized. Previous work from our laboratory demonstrated that TIL can organize in tertiary lymphoid structures (TLS) in BC-associated stroma. We further showed that CXCL13, produced by specialized CD4+ T follicular helper (TfhX13) cell, is an important TLS chemoattractant and associated with positive clinical outcomes. The current study investigated how immune cell functionally and regulation in TLS contributes to immune responses in BC. Methodology: We prospectively collected fresh primary BC tissues and prepared enzyme-free homogenates to produce TIL suspensions and tumor supernatants for flow cytometry and cytokine/chemokine/immunoglobulin (Ig) analysis, respectively. Matching formalin-fixed paraffin-embedded tumor tissues were analyzed using dual immunohistochemistry (IHC) and immunofluorescence (IF) confocal microscopy or multiplex IHC. Results: We show that CXCR5, the CXCL13 receptor, is expressed on subpopulations of CD4+ (Tfh) and CD8+ T cell TIL as well as the majority of TIL-B, with all CXCR5+ TIL co-localizing in TLS. The functional activities of Tfh TIL, evaluated using an in vitro assay with allogeneic human splenic B cells, reveals that PD-1hiICOS+ Tfh TIL (in some triple negative and HER2+ but not luminal BC) can provide help to TIL-B for Ig production. This observation is strengthened by additional data showing: 1) a correlation between functional (PD-1hiICOS+) Tfh TIL densities and IgG concentrations in primary BC supernatants; 2) a strong correlation between PD-1+ Tfh TIL and Ki67+ TIL-B in BC-associated TLS; and 3) cell-to-cell contact between Tfh TIL and TIL-B in TLS with active germinal centers. PD-1hiICOS+ (functional) and PD-1lo/intICOS− (non-functional) Tfh TIL were sorted for mRNA analysis with functional Tfh TIL expressing higher levels of IL-21, IFNγ and CXCL13. Higher IFNγ expression by PD-1hiICOS+ Tfh TIL suggests their functional Th1 orientation. CXCR5+ T follicular regulatory (Tfr) TIL were also detected in TLS and shown to express, CD25, demethylated FOXP3, and GARP, a marker of active TGFβ. Analyzing the ratio between Tfh and GARP+ Tfr TIL revealed that when the balance favors Tfh TIL, IgG production is increased. This Tfh/Tfr ratio was also correlated with activated CXCR5+CD8+ TIL in TLS. Multiplex IHC identified the positioning of CXCR5+ TIL subpopulations and demonstrated there are important cell-to-cell contacts between these subpopulations in active TLS. Conclusions: Our data show that Tfh TIL subpopulations play major roles in the functionality of BC-associated TLS. The balance between functionally active and regulatory CXCR5+ Tfh TIL together with active CXCR5+CD8+ TIL and CXCR5+ TIL-B appears to regulate immune activities in the tumor microenvironment. Tumors with functional Tfh TIL are linked to a more robust stromal and intratumoral infiltrate together with a ratio of active TLS >1. Thus, while TIL density scores provide important primary insight on immune activity in BC, their organization and subpopulation balances may offer key information for fine-tuning treatment selection options, particularly for immune-based therapies. Citation Format: Karen Willard-Gallo, Gregory Noel, Mireille Langouo Fontsa, Soizic Garaud, Alexandre de Wind, Gert Van den Eynden, Roberto Salgado, Anais Boisson, Celine Naveaux, Hugues Duvillier, Ligia Craciun, Martine Piccart-Gebhart, Denis Larsimont. Functional CXCR5+CD4+ follicular helper T cells in breast cancer associated tertiary lymphoid structures signal active immune responses at the tumor site [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-12.

Background Extracellular adenosine (Ado) is an immunosuppressive metabolite generated by the sequential activities of CD39 and CD73. High Ado, predominantly signaling through the A2AR, dampens immune responses and suppresses antitumor immunity. iTeos Therapeutics developed an A2AR antagonist, EOS100850, specifically designed to be a potent, highly selective and non-brain penetrant agent for treating a wide range of cancers. Methods The development of mIHC is a high-powered practical approach for phenotyping and correlating cellular interactions in the tumor microenvironment, which can also be used to monitor the response to treatment. We have optimized a 6-color mIHC panel for A2AR, CD39, CD73, TNAP, Cytokeratin and DAPI using tonsillar and breast cancer (BC) specimens using detection reagents. We also used an immune mIHC panel (CD4, CD8, CD20, CD68, FoxP3, Cytokeratin). Serial sections of tonsillar and BC tissues were stained and imaged at 20X on the Vectra Polaris (Akoya) platform and analyzed with InForm. Results Optimal dilutions for each antibody were first determined. A2AR staining was detected on immune cell subpopulations. CD39, CD73 and TNAP were expressed on immune cells and endothelial cells (EC). CD73 expression was elevated in Tertiary Lymphoid Structures. Then the order and pairing with an optimal fluorophore was examined. The relative positivity for the Ado pathway markers was accomplished first by full mIHC staining on tonsil tissues and subsequent validation on BC specimens. In BC tissues, A2AR, CD39 and TNAP expression was detected on various immune cell populations and EC. CD73 was additionally expressed by tumor cells. We also detected immune cells on BC tissue sections to characterize the cellular expression of Ado pathway markers, and to allow making correlations between expression of the Ado pathway markers and TIL infiltrate. Conclusion We have established a specific mIHC protocol to identify the Ado pathway in cancer specimens. Characterization of Ado pathway markers in conjunction with infiltrating immune subpopulations in matched pre- and post-therapy patient specimens will now be investigated to identify specific tumors and treatments that will most likely benefit from combination therapy with EOS100850. Legal entity responsible for the study iTeos Therapeutics. Funding iTeos Therapeutics. Disclosure A. Pabois: Full / Part-time employment: iTeos Therapeutics. V. Bodo: Shareholder / Stockholder / Stock options, Full / Part-time employment: iTeos Therapeutics. A. Boisson: Advisory / Consultancy: iTeos Therapeutics. S. Crosignani: Shareholder / Stockholder / Stock options, Full / Part-time employment: iTeos Therapeutics. O. De Henau: Full / Part-time employment: iTeos Therapeutics. M. Detheux: Shareholder / Stockholder / Stock options, Full / Part-time employment: iTeos Therapeutics. S. Garaud: Advisory / Consultancy: iTeos Therapeutics. J. Lager: Full / Part-time employment: iTeos Therapeutics. C. Martinoli: Full / Part-time employment: iTeos Therapeutics. M. Mercier: Full / Part-time employment: iTeos Therapeutics. C. Naveaux: Advisory / Consultancy: iTeos Therapeutics. N. Thomas: Advisory / Consultancy: iTeos Therapeutics. N. Wald: Full / Part-time employment: iTeos Therapeutics. A. Vezzu: Full / Part-time employment: iTeos Therapeutics. K. Willard-Gallo: Advisory / Consultancy: iTeos Therapeutics. E. Houthuys: Shareholder / Stockholder / Stock options, Full / Part-time employment: iTeos Therapeutics.

Background: Aging is a risk factor for developing malignant hemopathies including chronic lymphocytic leukemia (CLL). We evaluated the relevance of BACH2 and PRDM1 gene expression in the aging of immune cells and the immunodeficiency associated with CLL. Methods: Peripheral blood mononuclear cells were isolated from untreated CLL patients (n=41) and age-matched healthy donors (HD; n=60). T and B cells were purified (95%-99%) by magnetic isolation. BACH2, PRDM1, PD1, PDL1 and CDKN2A (p16INK4A) transcripts were quantified using RT-qPCR. ß-galactosidase activity was measured by flow cytometry. Cell apoptosis was analyzed after intracellular oxidative stress induced by etoposide treatment. ChIP-qPCR was done to find BACH2-apoptotic target genes in purified CD4+ T cells from HDs with/without etoposide treatment. Prognostic value of BACH2 and PRDM1 expression in purified CD19+ B cells was analyzed retrospectively in a cohort of CLL patients (n= 270), and correlated with clinical parameters. Results: BACH2 expression in HDs is significantly downregulated while PRDM1 expression increased in CD4+, CD8+ T and CD19+ B cells according to age groups. BACH2 expression is further reduced in T and B cells from CLL patients compared to age-matched HDs. Also, PRDM1 is significantly upregulated in T cells from CLL patients but not in leukemic B cells. PD1 expression is significantly upregulated in T cells in the older vs younger HDs and also in CLL patients compared with age-matched HDs. High PDL1 expression is also strongly correlated with increased age in HD B cells with a further increase detected in leukemic B cells. A strong inverse correlation was observed between BACH2 and PD1 in T cells; and between BACH2 and PDL1 in B cells from HDs. We also analyzed the link between BACH2 expression and senescence markers; CDKN2A and ß-galactosidase. CDKN2A expression inversely correlated with BACH2 in CD4+, CD8+ T and CD19+ B cells. ß-galactosidase activity showed an increase compared to BACH2 deficient lymphocytes in older compared to young HDs, suggesting that BACH2 deficiency leads to the accumulation of senescent cells. A strong correlation was observed between age-related BACH2 downregulation and a decrease in CD4+ T and CD19+ B cell apoptosis after etoposide treatment. ChIP-qPCR assay confirmed that BACH2 binds to genes involved in apoptosis and after etoposide treatment, BACH2 binding was repressed. In our retrospective cohort, increased BACH2 expression was correlated with improved treatment-free survival (p=0.0352). Conclusion: Decrease of BACH2 and increase of PRDM1, PD1 and PDL1 in T and B cells from CLL patients and aged-matched HDs, are significantly correlated with aging and could be part of immunosenescence. Involvement of BACH2 in resistance to drug-induced apoptosis of HD lymphocytes was documented and is currently investigated in the CLL cohort. Citation Format: Pushpamali De Silva, Vu Luan Dang Chi, Basile Stamatopoulos, Soizic Garaud, Vincent Thibaud, Hugues Duvillier, Jean-Nicolas Lodewyckx, Catherine Sibille, Karen Willard-Gallo, Dominique Bron. BACH2 and PRDM1 gene expression changes in T and B cells according to age of healthy individuals and significance in chronic lymphocytic leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3995.

Background: The level of immune suppression in an individual breast cancer (BC) patient is relevant for having a major benefit from treatments that act via or are directed to the immune response, such as anti-HER2 agents and immune checkpoint molecules, respectively. The link(s) between the tumor immune microenvironment and treatment responses has been investigated in HER2-positive BC. This study analyzed the association between baseline expression of genes regulating T cell activities (PD1, CTLA4, LAG3, TIM3, PDL1, PDL2, FOXP3, IL10, TGFβ (1-3), FOXP1 and other related genes) and pathologic complete response (pCR) rates in the NeoALTTO trial. Methods: NeoALTTO randomized early-stage HER2-positive BC patients to neoadjuvant trastuzumab (T), lapatinib (L) or T+L for 6 weeks followed by 12 weeks of concurrent paclitaxel. Anthracyclines were given after surgery (FEC). Patients with RNA sequencing data from their baseline tumor samples (N=254 patients out of 455 from the original cohort) were included in the current study. The primary endpoint, pCR, was defined as ypT0/is ypN0. Logistic regression was used for analysis of pCR. Cox regression univariate and multivariate (adjusted for clinicopathological parameters and treatment arms) analyses were performed. Results: In the total cohort analyzed, high PD1 (odds ratio, OR: 1.4, P=0.03), PDL2 (OR: 1.4, P=0.04), CTLA4 (OR: 1.4, P=0.03) and TGFβ3 (OR: 1.4, P=0.02) expression was associated with an increased probability of achieving pCR at the multivariate analysis. ERBB2 (HER2) expression was associated with pCR in the three arms (T: OR: 2.7, P=0.02; L: OR: 2.3, P=0.01; T+L: OR: 5, P Citation Format: Cinzia Solinas, Pushpamali De Silva, David Venet, Soizic Garaud, Chunyan Gu-Trantien, Florentine Hilbers, Evandro de Azambuja, Olena Werner, Lorena De la Peña, Amylou Dueck, Serena Di Cosimo, Istvan Lang, Jens Huober, Sherko Küemmel, Carsten Denkert, Roberto Salgado, Christos Sotiriou, Martine Piccart-Gebhart, Debora Fumagalli, Karen Willard-Gallo. Immune regulatory gene expression and clinical outcome in the NeoALTTO trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3132.

The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by recent large clinical trials (presented at SABCS 2013). The relationship between protective immunity, observed in some patients, and critical features of lymphoid subset composition and organization remain unknown. Our recent work revealed that tertiary lymphoid structures (TLS), principally composed of T cells, B cells and dendritic cells, are present in the peri-tumoral regions of breast tumors and associated with a CD4+ follicular helper T (Tfh) cell presence. Through retrospective analyses, we determined that TLS were associated with good clinical outcomes in both the neo-adjuvant and adjuvant settings. To gain insight into the immune components linked with a significant TIL and TLS presence, we initiated a prospective flow cytometric study. We systematically immunophenotyped T and B cell TIL in breast tissues from tumors (n=125), non-adjacent non-tumor tissues (NANT, n=115) and normal tissue from mammary reductions (n=26) on the day of surgery. TIL organization and spatial distribution was subsequently analyzed by immunohistochemistry (IHC) and immunofluorescence (IF) on paraffin sections for a subset of patients (n=78). The fresh tissue analyses revealed that TIL density was a continuum across the 125 patients analyzed. A cutoff for TIL-positive and -negative tumors was set at 58 CD45+ TIL per mm3 of tissue based on the number of CD45+ cells present in the remarkably similar normal and NANT tissues. Applying this threshold to BC, 65% were TIL-positive with approximately one-third considered extensively infiltrated. TIL-positive tumors are characterized by an increase in CD4+ T cells and CD19+/CD20+ B cells. The median CD4/CD8 ratio was >1 in TIL-positive compared to 95% of CD45+ cells) IHC and scored by trained pathologists. The best correlation was observed between CD3/CD20 flow cytometry and CD3/CD19 IHC, the latter also associated with lower inter-observer variability. TIL density was positively correlated with proliferation (Ki67 & histological grade) and hormone receptor negativity while TLS were more frequent in younger women. These data suggest that organized immune responses in TLS adjacent to the tumor bed provide an effective location for generating anti-tumor memory T and B cell responses. Citation Format: Karen Willard-Gallo, Laurence Buisseret, Soizic Garaud, Chunyan Gu-Trantien, Alexandre de Wind, Sébastien Duquenne, Denis Larsimont, Christos Sotiriou, Martine Piccart. The significance of tumor infiltrating lymphocyte density, subset composition and organization in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-3.

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 (R2=0.74) but this did not extend to tertiary lymphoid structures (R2=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.

BACKGROUND The tumor immune response is increasingly associated with better clinical outcomes in breast and other cancers. However, the evaluation of tumor-infiltrating lymphocytes (TILs) relies on histopathological measurements with limited accuracy and reproducibility. Here, we profiled DNA methylation markers to identify a methylation of TIL (MeTIL) signature that recapitulates TIL evaluations and their prognostic value for long-term outcomes in breast cancer (BC). METHODS MeTIL signature scores were correlated with clinical endpoints reflecting overall or disease-free survival and a pathologic complete response to preoperative anthracycline therapy in 3 BC cohorts from the Jules Bordet Institute in Brussels and in other cancer types from The Cancer Genome Atlas. RESULTS The MeTIL signature measured TIL distributions in a sensitive manner and predicted survival and response to chemotherapy in BC better than did histopathological assessment of TILs or gene expression-based immune markers, respectively. The MeTIL signature also improved the prediction of survival in other malignancies, including melanoma and lung cancer. Furthermore, the MeTIL signature predicted differences in survival for malignancies in which TILs were not known to have a prognostic value. Finally, we showed that MeTIL markers can be determined by bisulfite pyrosequencing of small amounts of DNA from formalin-fixed, paraffin-embedded tumor tissue, supporting clinical applications for this methodology. CONCLUSIONS This study highlights the power of DNA methylation to evaluate tumor immune responses and the potential of this approach to improve the diagnosis and treatment of breast and other cancers. FUNDING This work was funded by the Fonds National de la Recherche Scientifique (FNRS) and Televie, the INNOVIRIS Brussels Region BRUBREAST Project, the IUAP P7/03 program, the Belgian "Foundation against Cancer," the Breast Cancer Research Foundation (BCRF), and the Fonds Gaston Ithier.

Background: CD73 is an ecto-enzyme that promotes tumor immune escape through the production of immunosuppressive extracellular adenosine in the tumor microenvironment. Several CD73 inhibitors and adenosine receptor antagonists are being evaluated in phase I clinical trials. Objective: To investigate the prognosis significance of CD73 in human triple-negative breast cancer. Design and setting: This is a prospective-retrospective biomarker analysis. Using multiplex immunofluorescence and image analysis, we assessed CD73 protein expression on tumor cells, tumor-infiltrating leukocytes and stromal cells on full-face sections from formalin-fixed paraffin-embedded primary breast tumors. Participants: 122 samples of triple-negative breast cancer from the BIG 02-98 adjuvant phase III clinical trial were included in our analysis. This trial compared the addition of taxanes to anthracyclines-based chemotherapy in node-positive breast cancer. Results: Our results demonstrated that high levels of CD73 expression on epithelial tumor cells were significantly associated with reduced disease-free survival (DFS) and overall survival (OS) in patients with triple-negative breast cancer. Using the median as a threshold between low and high levels of CD73 on epithelial cells, hazard ratios (HR) adjusted for grade, number of positive lymph nodes and tumor size, were of 2.21 (95% confidence interval (CI): 1.15-4.25); p=0.02 for DFS and of 2.47 (95%CI: 1.21-5.07); p=0.01 for OS. CD73 expression negatively correlated with tumor immune infiltration (Spearman's R= -0.50, p Conclusion and relevance: Taken together, our study provides further support that CD73 expression is associated with a poor prognosis and reduced anti-tumor immunity in human triple-negative breast cancer and that targeting CD73 could be a promising strategy to reprogram the tumor microenvironment in this breast cancer subtype. Citation Format: Buisseret L, Pommey S, Allard B, Garaud S, Bergeron MA, Cousineau I, Ameye L, Paesmans M, Crown JPA, Di Leo A, Piccart-Gebhart M, Willard-Gallo K, Sotiriou C, Stagg J. Clinical significance of CD73 expression in triple-negative breast cancer from the BIG 02-98 adjuvant phase III clinical trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-07.

B lymphocytes from chronic lymphocytic leukemia (CLL) display some CD5 transcripts for CD5 containing the known exon 1 (E1A) and other CD5 transcripts containing the new exon 1 (E1B). These malignant B cells, as well as B cell lines transfected with cDNA for E1A-cd5 or with cDNA for E1B-cd5 produce IL-10, raising the possibility that CD5 participates in the secretion of IL-10. We identified transcription factors involved in this production in CD5+ B lymphocytes from CLL patients and in E1A-cd5–transfected or E1B-cd5–transfected Jok cells. STAT3 is activated via phosphorylation of serine 727 but also NFAT2 through its translocation into the nucleus. Chromatin immunoprecipitation experiments confirmed the role of STAT3 and allowed the discovery of a role for NFAT2 in IL-10 production. Both transcription factors bind not only to the enhancer of the Il-10 gene but also to the promoter of the Il-5 and Il-13 genes. Furthermore, transfection of B cell lines with E1A-cd5 or E1B-cd5 established that activation of STAT3 and NFAT2 is regulated by CD5. The same holds true for the production of IL-10, IL-5, and IL-13 and the expression of the receptors for these cytokines. This interpretation was confirmed by two experiments. In the first, downregulation of CD5 by small interfering RNAs lowered the production of IL-10. In the second experiment, transfection of the GFP-NFAT2 gene into B lymphocytes induced nuclear translocation of NFAT2 in CD5+ but not in CD5− B cells. Thus, CD5 expression is associated with NFAT2 activity (and mildly STAT3 activity), indicating that CD5 controls IL-10 secretion.