Search

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

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.

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.

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.

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.

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 FOXP1hi and FOXP1lo primary BC. Finding: FOXP1 expression was higher in estrogen receptor positive compared to negative BC. FOXP1hi tumors were significantly associated with lower TIL and fewer tertiary lymphoid structures (TLS) compared to FOXP1lo 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 FOXP1hi compared to FOXP1lo primary BC. Lymphocyte migration using primary BC supernatants detected decreased mobility toward FOXP1hi supernatants. FOXP1lo 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., SCOPUS: ar.j, info:eu-repo/semantics/published

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

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.

The forkhead box P1 (FOXP1) transcription factor has been shown to regulate the generation and maintenance of quiescent naive murine T cells. In humans, FOXP1 expression has been correlated with overall survival in patients with peripheral T-cell lymphoma (PTCL), although its regulatory role in T-cell function is currently unknown. We found that FOXP1 is normally expressed in all human leukocyte subpopulations. Focusing on primary human CD4+ T cells, we show that nuclear expression of FOXP1 predominates in naive cells with significant downregulation detected in memory cells from blood and tonsils. FOXP1 is repressed following in vitro T-cell activation of naive T cells, and later re-established in memory CD4+ T cells, albeit at lower levels. DNA methylation analysis revealed that epigenetic mechanisms participate in regulating the human FOXP1 gene. ShRNA-mediated FOXP1 repression induces CD4+ T cells to enter the cell cycle, acquire memory-like markers and upregulate helper T-cell differentiation genes. In patients with lymphoproliferative disorders, FOXP1 expression is constitutionally repressed in the clonal T cells in parallel with overexpression of helper T-cell differentiation genes. Collectively, these data identify FOXP1 as an essential transcriptional regulator for primary human CD4+ T cells and suggest its potential important role in the development of PTCL.

Age-related genetic changes in lymphocyte subsets are not currently well documented. BACH2 is a transcription factor that plays an important role in immune-mediated homeostasis by tightly regulating PRDM1 expression in both B-cells and T-cells. BACH2 gene expression is highly sensitive to DNA damage in aged mice. This concept led us to investigate the variation in BACH2 and also PRDM1 expression in major lymphocyte subsets with age., info:eu-repo/semantics/published

Tumor-infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in numerous large clinical trials for HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC). Our previous work in human demonstrated that TIL can organize in tertiary lymphoid structures (TLS) with the B-cell chemoattractant, CXCL13, playing a key role in their formation as well as having a strong association with positive clinical outcomes. The present study investigated how TLS functionally contribute to immune responses in human BC. We prospectively collected fresh primary human BC tissues and prepared enzyme-free homogenates to produce TIL suspensions and tumor supernatants for flow cytometry and cytokine/chemokine/immunoglobulin (Ig) quantification, respectively. Matching formalin-fixed, paraffin-embedded tumor tissues were used for spatial analysis by dual immunohistochemistry and immunofluorescent confocal microscopy. We show that CD3+CD4+, CD3+CD8+ and CD19+ TIL in human BC contain subpopulations expressing CXCR5 (the CXCL13 receptor) and are co-localized in TLS. An in vitro functional assay activating Tfh cells with allogeneic B cells from human spleen evaluated the functionality of human BC Tfh TIL (CD45+CD3+CD4+CXCR5+). These experiments reveal that ICOS+PD-1hi Tfh TIL from TN and HER2+, but not luminal, BC possess helper activities for B cell IgG production. This observation is supported by a correlation between ICOS+PD-1hi Tfh TIL densities and IgG concentrations in primary human BC supernatants. A comparison of ICOS+PD-1hi and ICOS-PD-1- Tfh TIL confirms the activated, functional nature of these specialized TIL, which are characterized by high IL-21, IL-10 and CXCL13 mRNA expression. High IFNγ mRNA expression was detected in ICOS+PD-1hi Tfh TIL, suggesting their functional Th1 orientation. We also identified follicular regulatory T cells (Tfr), located in TLS that express CXCR5, CD25, demethylated Foxp3 and GARP, a marker of TGFβ activity. Tfr regulation of Tfh functions were evaluated by determining the ratio between functional Tfh TIL and GARP+ Tfr TIL. This analysis reveals a strong correlation between the Tfh/Tfr ratio and IgG production in primary tumor supernatants from TN and HER2+ human BC. In addition, strong correlations were observed with IgA and IgM production in HER2+ human BC. Our data show that Tfh TIL in human BC are major contributors to TLS activity, with the ratio between functional Tfh TIL and activated Tfr TIL directly influencing Ig production by B cells. Effective antitumor immunity in BC is therefore not only characterized by TIL density and organization but also by the balance between effector and regulatory TIL. These findings constitute important elements to consider when choosing patients for immunotherapy. Citation Format: Grégory Noël, Mireille Langouo, Soizic Garaud, Anaïs Boisson, Hugues Duvillier, Gert Van den Eynden, Denis Larsimont, Karen Willard-Gallo. The balance between activated follicular helper T cells and follicular regulatory T cells within tertiary lymphoid structures guides antitumor immune responses in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A40.

Interest is growing 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), confirms 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 Igs in 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 microdissected 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. This abstract is also being presented as Poster B99. Citation Format: Pushpamali De Silva, Soizic Garaud, Cinzia Solinas, Grégory Noël, Mireille Langouo Fontsa, Anaïs Boisson, Alexandre de Wind, David Venet, Gert Van den Eynden, Hugues Duvillier, Céline Naveaux, Ligia Craciun, Dominique Bron, Martine Piccart-Gebhart, Denis Larsimont, Karen Willard-Gallo. Active and quiescent tertiary lymphoid structures, differentiated using FOXP1 expression, play a role in immunity to breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR10.

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

This study investigated the prevalence of TIL subpopulations, TLS, PD-1 and PD-L1 in tumors from TNBC patients harboring wild-type or mutated BRCA1 or BRCA2 germline genes. This TNBC cohort included 85% TIL-positive (≥10%) tumors with 21% classified as TILhi (≥50%). Interestingly, the BRCAmut group had a significantly higher incidence of TILpos tumors compared to the BRCAwt group (P = 0.037). T cells were dominant in the infiltrate but no statistically significant differences were detected between BRCAwt and BRCAmut for CD3+, CD4+ and CD8+ T cells or CD20+ B cells. TLS were detected in 74% of tumors but again no significant differences between the BRCA groups. PD-1 expression was observed in 33% and PD-L1 in 53% (any cell, cut-off ≥1%) tumors for the entire TNBC cohort. PD-1 expression correlated with PD-L1 and both with TIL and TLS but was not associated with BRCA mutational status. Our analyses reveal that BRCAwt and BRCAmut TNBC are similar except for a significant increase of TILpos tumors in the BRCAmut group. While BRCA gene mutations may not directly drive immune infiltration, the greater number of TILpos tumors could signal greater immunogenicity in this group.

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.

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.

It is now widely accepted that the immune microenvironment of tumors and more precisely Tumor Infiltrating Lymphocytes (TIL) play an important role in cancer development and outcome. TILs are considered to be important prognostic and predictive factors based on a growing body of clinical evidence; however, their presence at the tumor site is not currently assessed routinely. FTIR (Fourier transform infrared) imaging has proven it has value in studying a range of tumors, particularly for characterizing tumor cells. Currently, very little is known about the potential for FTIR imaging to characterize TIL. The present proof of concept study investigates the ability of FTIR imaging to identify the principal lymphocyte subpopulations present in human peripheral blood (PB). A negative cell isolation method was employed to select pure, label-free, helper T cells (CD4(+)), cytotoxic T cells (CD8(+)) and B cells (CD19(+)) from six healthy donors PB by Fluorescence Activated Cell Sorting (FACS). Cells were centrifuged onto Barium Fluoride windows and ten infrared images were recorded for each lymphocyte subpopulation from all six donors. After spectral pre-treatment, statistical analyses were performed. Unsupervised Principal Component Analyses (PCA) revealed that in the absence of donor variability, CD4(+) T cells, CD8(+) T cells and B cells each display distinct IR spectral features. Supervised Partial Least Square Discriminant Analyses (PLS-DA) demonstrated that the differences between the three lymphocyte subpopulations are reflected in their IR spectra, permitting their individual identification even when significant donor variability is present. Our results also show that a distinct spectral signature is associated with antibody binding. To our knowledge this is the first study reporting that FTIR imaging can effectively identify T and B lymphocytes and differentiate helper T cells from cytotoxic T cells. This proof of concept study demonstrates that FTIR imaging is a reliable tool for the identification of lymphocyte subpopulations and has the potential for use in characterizing TIL.

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

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., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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.

International audience; CD5(+) B lymphocytes have distinct functional properties compared with B lymphocytes that lack CD5. However, it remains unclear if and how the CD5 molecule modulates B lymphocyte biology and responses. Our recent studies have revealed that CD5 promotes constitutive activation of multiple signaling pathways including extracellular signal-regulated kinases (ERK1/2), phosphatidylinositol 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) and calcineurin-NFAT signaling pathways. Further, changes in cytokine production including the production of IL-10 are related to the activation of the transcription factors NFAT2 and STAT3. All in all, these studies provide a framework for understanding how CD5 impacts B lymphocyte biology and responses.

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.

Reproducible evaluation of immune infiltration parameters is needed for the development of reliable biomarkers in the era of cancer immunotherapy. This study investigated inter-observer reproducibility when evaluating tumor-infiltrating lymphocytes (TIL), tertiary lymphoid structures (TLS), PD-1 and PD-L1 positivity on immunohistochemically-stained (IHC) breast cancer (BC) tissues, including detailed analysis of spatial positioning in the tumor microenvironment and expression on individual positive cells. Retrospectively collected archival BC samples (N=441) were dual IHC-stained for CD3 (pan T cells) plus CD20 (pan B cells) for TIL and TLS assessment with a consecutive tissue section dual-IHC stained for PD-1 and PD-L1. The 882 tissue sections were independently read by two immuno-pathologists blinded to the clinical data for specific studies between 2014 and 2017. Five independent study series were used for the current analysis: 1) samples of BC relapses, 2) residual disease in breast after neodjuvant treatment, and 3) three independent series of untreated primary tumors. Intraclass correlation coefficients for evaluations are shown in the table below: RelapsesResidual diseaseUntreated primary tumorsSeries 1: N=88Series 2: N=20Series 3: N=85Series 4: N=113Series 5: N=135201420142014-201520162017ICCICCICCICCICC%Stromal TIL0.85 [0.76-0.90]0.71 [0.27-0.89]0.79 [0.68-0.87]0.74 [0.62-0.82]0.77 [0.68-0.83]%Intratumoral TIL0.40 [0.09-0.61]0.63 [0.08-0.86]0.73 [0.58-0.82]0.87 [0.81-0.91]0.64 [0.51-0.74]%Global TIL0.87 [0.80-0.92]0.79 [0.47-0.92]0.83 [0.74-0.89]0.80 [0.72-0.86]0.81 [0.73-0.86]%CD30.83 [0.74-0.89]0.71 [0.28-0.89]0.81 [0.70-0.88]0.72 [0.60-0.80]0.73 [0.63-0.81]%CD200.74 [0.62-0.81]0.81 [0.52-0.92]0.76 [0.63-0.84]0.78 [0.68-0.84]0.78 [0.70-0.84]TLS0.80 [0.70-0.87]0.78 [0.45-0.91]0.83 [0.74-0.89]0.95 [0.93-0.97]0.97 [0.96-0.98]%PD-L1 tumor cells0.78 [0.66-0.86]0.56 [-0.12-0.82]0.82 [0.73-0.89]0.62 [0.45-0.74]0.45 [0.25-0.61]%PD-L1 stromal cells0.61 [0.40-0.74]-1.56 [-1.53-0.60]0.43 [0.12-0.63]0.61 [0.44-0.73]0.31 [0.05-0.5]%PD-L1 immune cells0.53 [0.29-0.70]0.94 [0.84-0.97]0.29 [-0.09-0.54]0.38 [0.10-0.57]0.10 [-0.25-0.35]%PD-1 TLS-0.00 [-0.54-0.35]NA0.72 [0.57-0.82]0.57 [0.35-0.72]0.76 [0.62-0.84] Cohen's κ coefficients reveal poor agreement (κ1%). Sporadically, fair agreement (κ>0.4) was observed for PD-L1 expressed by immune and tumor cells. Globally, the inter-observer agreement remained constant overtime. Poor reproducibility for categorical global PD-1 and global PD-L1 assessment was observed. On the contrary, pathologists have excellent reproducibility for assessing global TIL and TLS, good/excellent reproducibility for PD-1 in TLS (primary tumors) and for PD-L1 expression on tumor cells which make these of great interest for clinical use. Citation Format: Cinzia Solinas, Gert Van den Eynden, Alexandre De Wind, François Richard, Anaïs Boisson, Soizic Garaud, Karen Willard-Gallo. Reliability of immune biomarker assessment in breast cancer: A report on interobserver variability from studies at a single institution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1624.

Introduction: High levels of tumor infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in patients with HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC) . Recently, we demonstrated that 60% of BC TIL are organized in tertiary lymphoid structures (TLS) located in the stroma. We further identified a CXCL13-producing CD4+ T follicular helper cell (Tfh) subpopulation and demonstrated that this chemokine, important for TLS formation, is associated with positive clinical outcomes in BC. The aim of the present study was to investigate how conventional CD4+ Tfh cells, expressing the CXCL13 receptor CXCR5, contribute to immune function and regulation in BC-associated TLS. Methods: We prospectively collected fresh primary BC tissues and prepared enzyme-free homogenates to prepare tumor supernatant and TIL for flow cytometric analysis and sorting. Matching formalin-fixed paraffin-embedded (FFPE) were used for subsequent spatial analysis by dual IHC and confocal microscopy. Results: Flow cytometric analyses show that ~15% CD4+, ~13% CD8+ and >95% B cell TIL express CXCR5 while confocal microscopy reveals that these CXCR5+ TIL subpopulations co-localize in BC TLS and their presence is tightly correlated. BC Tfh TIL (compared to activated tonsillar Tfh) have a memory phenotype and express ICOS and PD-1 suggesting they are activated, but BCL6 is undetectable and CXCL13 positive cells are rare in only some tumors. RNA analysis detected high expression of IL-21, IL-10 and CXCL13 in Tfh TIL. Investigation into the role of Tfh cells in TLS functionality was accomplished via an in vitro assay where Tfh TIL were activated with allogeneic splenic B cells. These experiments reveal that only ICOS+PD-1+Tfh TIL from TN/HER2+ BC are capable of inducing IgM and IgG secretion by B cells. A comparison of ICOS+PD-1+ with ICOS-PD-1- Tfh TIL confirms an activated, functional nature for the former characterized by high levels of IL-21, IL-10 and CXCL13 mRNA expression. We also found specialized follicular regulatory T cells (Tfr), expressing CXCR5, CD25, demethylated Foxp3 and GARP, localized in TLS. To understand the regulation of Tfh function(s) by Tfr we quantified immunoglobulins in the primary tumor supernatant, finding a correlation between the Tfh/Tfr ratio (fresh tissues) and IgG and IgM production (supernatant) in TN/HER2+ BC. These data suggest that the balance between effector and regulatory Tfh influences B cell differentiation in TLS. Conclusions: We show that it is possible to isolate functional Tfh TIL from fresh BC tissues, demonstrate their activation and functional state and link a Tfr presence with negative regulation of TLS function. These data begin to shed light on anti-tumor immune responses occurring in TLS at the tumor site, whose functional activities may have important treatment implications, particularly for immunotherapy in BC. Citation Format: Mireille Langouo, Gregory Noël, Gert Van den Eynden, Alexandre De Wind, Soizic Garaud, Pushpamali De Silva, Cinzia Solinas, Anais Boisson, Celine Naveaux, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo. Immune functions and regulation of follicular helper CD4+CXCR5+T cells in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4689.

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 the etiology of BRCA1/2 related BC. Here, we present the primary results of D-BEYOND, a window of opportunity 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. Blood, tumor and normal adjacent breast tissue were collected at baseline and at surgery. The primary endpoint was geometric mean change in tumor Ki67 assessed by immunohistochemistry (IHC) from baseline to surgery. Absolute Ki67 responders were defined as having 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 sRANKL decreased in all patients (P < 0.001) whereas serum levels of 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 change after treatment; 0.98, 95% CI 0.76-1.26; P = 0.90) and there were no absolute Ki67 responders. Twenty-four pre- and post-treatment tumor pairs were available for RANK/L staining and TILs assessment. There was no significant difference in RANKL and RANK H-score in tumors after treatment (P = 0.842, P = 0.142, respectively) but we observed a decrease of RANKL H-score in 3 tumors (12.5%) and an increase of RANK H-score in 5 tumors (20%). Interestingly, there was a significant increase in the percentage of stromal TILs after treatment (geometric mean change of 2.51, 95% CI 1.58-3.97; P = 0.004). There were 10/24 patients (41.7%) with a change in TILs of at least 10%, all of them having an increase in TILs presence (P = 0.002). Conclusion Short course of denosumab did not reduce tumor proliferation rate. However, it induced a significant increase in TILs. These findings suggest that denosumab may potentiate immunotherapy efficacy, at least in young BC patients. Additional results including immune cell profiling by multiplex IHC and RNA-sequencing of tumor and normal tissues will be presented at the meeting. Citation Format: Bastien Nguyen, Marion Maetens, Roberto Salgado, David Venet, Peter Vuylsteke, Laura Polastro, Hans Wieldiers, Philippe Simon, Geoff Lindeman, Denis Larsimont, Gert Van den Eynden, Chloe Velghe, Francoise Rothe, Soizic Garaud, Stefan Michiels, Karen Willard-gallo, Hatem A. Azim, Sherene Loi, Martine Piccart, Christos Sotiriou. D-BEYOND: A window of opportunity trial evaluating denosumab, a RANK-ligand (RANKL) inhibitor and its biological effects in young pre-menopausal women diagnosed with early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT101.

B lymphocytes are divided into two subpopulations, B1 and B2 cells based on expression of the T cell-associated protein CD5. Natural B1 cells are further divided into B1a cells that express CD5 on their membrane and B1b cells that do not but share most other biological characteristics of B1a cells. Recent studies from our laboratory have revealed, in humans, the existence of two alternative isoforms of the CD5 protein. A cell surface CD5 isoform which uses exon 1A (E1A) of the gene in B1a cells, and an intracellular isoform which uses exon 1B (E1B) mainly in human B1b cells. Indeed, the protein isoform encoded by transcripts containing E1B lack the leader peptide and is, thus, retained in the cytoplasm of B cells. The restriction of interleukin (IL)-10 to B1 lymphocytes in the mouse raises the possibility that the human CD5-E1B-expressing B cells produce IL-10. This prediction was confirmed in the CD5 negative Jok-1 B cells transfected with cDNA for either isoforms resulted in high level IL-10 production. Our data indicate that E1B-CD5-expressing B cells have the capacity to interfere with the immune response through their ability to produce high levels of IL-10.

Defective regulation of secondary immunoglobulin V(D)J gene rearrangement promotes the production of autoantibodies in systemic lupus erythematosus (SLE). It remains unclear, however, whether the regulation of the recombination-activating genes RAG1 and RAG2 is effective in SLE. RAG1 and RAG2 messenger RNA expression was analysed before and after in vitro activation of sorted CD19(+) CD5(-) B cells with anti-immunoglobulin M antibodies, in 20 SLE patients and 17 healthy controls. The expression of CDK2 and p27(Kip1) regulators of the RAG2 protein, were examined. The levels of interleukin-6 (IL-6) and its influence on RAG regulation were also evaluated in vitro. SLE patients had increased frequency of RAG-positive B cells. B-cell receptor (BCR) engagement induced a shift in the frequency of kappa- and lambda-positive cells, associated with a persistence of RAG messenger RNA and the maintenance of RAG2 protein within the nucleus. While expression of the RAG2-negative regulator CDK2 was normal, the positive regulator p27(Kip1) was up-regulated and enhanced by BCR engagement. This effect was the result of the aberrant production of IL-6 by SLE B cells. Furthermore, IL-6 receptor blockade led to a reduction in p27(Kip1) expression, and allowed the translocation of RAG2 from the nucleus to the cytoplasm. Our study indicates that aberrant production of IL-6 contributes to the inability of SLE B cells to terminate RAG protein production. Therefore, we hypothesize that because of constitutive IL-6 signalling in association with BCR engagement, SLE B cells would become prone to secondary immunoglobulin gene rearrangements and autoantibody production.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

Breast cancer (BC) development and progression is influenced by the intrinsic properties of tumor cells as well as their surrounding microenvironment. Cells found in the microenvironment include stroma cells, fibroblasts, endothelial cells, pericytes, adipocytes and various types of infiltrating immune cells. The immune infiltrate generally includes myeloid cells and lymphocytes (tumor infiltrating lymphocytes (TILs)) composed of B cells, T cells and natural killer cells. Recently, the immune response has been named as a new hallmark of cancer. In BC, the extent of lymphocyte infiltration is predictive for better local responses to neoadjuvant chemotherapy and prognostic for long-term disease-free survival.1, 2, 3 However, little is known about the processes involved in attracting specific subsets of TIL to the tumor microenvironment.

Patient outcomes have been linked to the presence of tumor infiltrating lymphocytes (TIL) in solid tumors. In human breast cancer (BC), higher TIL infiltration is associated with a better prognosis and also predicts relevant responses to pre-operative chemotherapy. TIL are primarily composed of T cells, albeit around 20% of BC patients show significant B cell infiltration, and can organize in tertiary lymphoid structures (TLS) located in the peritumoral stroma 1, which are associated with survival in HER2+ and triple negative BC patients. Further, these studies revealed that CD4+ follicular helper T (Tfh) cells producing CXCL13 were specifically associated with peritumoral TLS. CXCL13 is an important B cell chemoattractant whose function is to recruit B cells to the germinal center (GC) in secondary lymphoid organs and TLS, where they can mature and differentiate into memory or antibody-producing B cells. Our recent efforts have focused on exploring the role of Tfh, B cells and CXCL13 play in the development and/or maintenance of GC-like structures in BC-associated TLS. We first derived a GC-B cell gene signature for integration in our published Tfh cell gene signature 1. The combined gene signature was tested for its ability to sensitively detect BC-associated TLS using a qRT-PCR-based assay and a retrospective series (n=54) of formalin-fixed paraffin-embedded (FFPE) BC tissues. These data revealed a correlation between gene signature expression and the extent of TIL and TLS scored by trained pathologists on dual-immunohistochemistry stained (CD3+CD20 for T and B cells, respectively) FFPE tissue sections. The detection of TLS using our combined GC-B cell/Tfh cell gene signature was subsequently confirmed using tissues from our prospective BC cohort (n=83). In addition, CXCL13 gene expression was well correlated with genes associated with GC-B cells and Tfh, indicating these parameters are closely related, as confirmed by immunofluorescence staining on FFPE tissues. Further understanding the factors that promote TLS formation in vivo could provide important insight for treatment decisions in BC. CXCL13 expression was originally identified as an important signal associated with TLS that was predictive for patient outcomes 1. Factors capable of inducing CXCL13 expression in CD4+ T cells isolated from peripheral blood were investigated using flow cytometry. TGFβ1 alone or together with several cytokines (IL4, IL12, IL21, IL23 and in particular IL2 blockade) increased CXCL13 expression in activated CD4+ T cells. Similar to our characterization of Tfh TIL in fresh tumor tissues, these CXCL13-producing CD4+ T cells were CXCR5 negative and expressed the Tfh marker ICOS; however, they only had low levels of PD-1 expression compared to PD-1hi Tfh cells. CD8+ T cells were also found to produce CXCL13 albeit at low levels. The currently ongoing identification of critical genes involved in regulating CXCL13 production in treated CD4+T cells will help to elucidate the mechanism(s) underlying chemokine induction. The increased accuracy in TIL and TLS detection in BC together with a better understanding of the role Tfh and CXCL13 play in these structures (and GC) development should help to identify the critical immune components involved in BC TLS formation. 1Gu-Trantien et al. J Clin Invest. 2013 Citation Format: Edoardo Migliori, Chunyan Gu-Trantien, Soizic Garaud, Gert Van den Eynden, Alexandre De Wind, Pushpamali De Silva, Cinzia Solinas, Anais Boisson, Celine Naveaux, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo. Investigating the role of follicular helper T cells, B cells and CXCL13 in breast cancer-associated tertiary lymphoid structures. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A62.

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.

Background: Aging is characterized by a progressive decline in immune surveillance that favors tumor development in older patients. One mechanism used by pre-malignant and/or malignant cells to escape immune surveillance is the upregulation of inhibitory immune checkpoint molecules, such as PD-1 and PD-L1. Another process associated with aging is genetic or epigenetic modifications of tumor suppressor genes (TSGs). We have reported a correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate TSG(1). We thus examined - on a molecular level - the expression of specific transcription factors (BACH2 and PRDM1) and checkpoint molecules (PD-1 and PD-L1) in the major lymphocytes subsets for their potential as predictive markers of immunosenescence. Methods: Lymphocyte subpopulations (CD3+/CD4+; CD3+/CD8+ and CD19+) were isolated for subsequent molecular analyses using the MACS technology (Miltenyi), with the purity of each lymphocyte subpopulation between 95%-99%. PD-1 (PDCD1), PD-L1 (CD274), IL4, IFNG, BACH2 and PRDM1 (Blimp1) mRNA transcripts were quantified in the purified subpopulations using qRT-PCR. BACH2 and PRDM1 protein expression were examined by Western blotting. Peripheral blood mononuclear cells were treated by Etoposide (VP16) to induce intracellular oxidative stress. Apoptotic function was assessed by flow cytometry (Annexin-V and Propidium Iodide). Results: Peripheral blood samples were obtained from 40 healthy donors (HDs) between the ages of 20 to 90 yrs, subdivided into BACH2-deficient mice have been shown to have increased numbers of IL4-producing CD4+ T cells after activation(2), with BACH2 known to repress PRDM1 expression in B andT cells(2,3). We, therefore, examined BACH2 gene expression in the HD groups finding it was significantly downregulated in CD4+, CD8+ T cells and CD19+ B cells from the older HD group (p=0.001; 0.004 and 0.03, respectively). BACH2 expression was further reduced in CD4+, CD8+ T cells and CD19+ B cells from CLL patients compared to HD of similar age (p=0.001; PD-1 gene expression was significantly upregulated in CD4+, CD8+ T cells (p=0.015 and 0.02) in the older HD group. PD-1 was also significantly upregulated in the same T cells subpopulations isolated from untreated CLL patients (p=0.001 and 0.002) when compared to a similar HD age group. High PD-L1 gene expression was correlated with increased age in HD B cells (p=0.04) with a further increase detected in leukemic B cells compared with the older HD group (p=0.001). We also observed an inverse correlation between BACH2 and PD-1 in CD4+, CD8+ T cells (r=0.59 and 0.68); and between BACH2 and PD-L1 in CD19+ B cells (r=0.66). We further examined VP16-induced apoptosis in isolated lymphocytes subpopulations. As recently reported in BACH2-deficient mice(4), our observation showed that BACH2 downregulation was strongly correlated with resistance to apoptosis in CD4+, CD8+ T cells and leukemic B cells (r=0.61; 0.75 and 0.69, respectively). Conclusion: These data suggest that BACH2, PRDM1, PD-1 and PD-L1 gene expression is correlated with aging and age-related immunosuppression. These effects are even more pronounced in leukemic cells from CLL patients. 1. C. Sibille, et al.,(personal data) ; 2. S. Tsukumo, et al. Proc. Natl Acad Sci USA. 2013 ; 3. H. Tanaka et al. J Biol Chem. 2016 ; 4. Muto, A., et al. J Biol Chem. 2002; Disclosures No relevant conflicts of interest to declare.

International audience; Although not exclusive, mounting evidence supports the fact that DNA methylation at CpG dinucleotides controls B-cell development and the progressive eliminati or inactivation of autoreactive B cell. Indeed, the expression of different B ce specific factors, including Pax5, rearrangement of the B-cell receptor (BCR) and cytokine production are tightly controlled by DNA methylation. Among normal B cells, the autoreactive CD5+ B cell sub-population presents a reduced capacity to methylate its DNA that leads to the expression of normally repressed genes, such as the human endogenous retrovirus (HERV). In systemic lupus erythematosus (SLE) patients, the archetype ofautoimmune disease, autoreactive B cells are characterized by their inability to induce DNA methylation that prolongs their survival. Finally, treating B cells with demethylating drugs increased their autoreactivity. Altogether this suggests that a deeper comprehension ofDNA methylation in B cells may offer opportunities to develop new therapeutics to control autoreactive B cells.

International audience; Autoreactive B cells are central in the pathogenesis of autoimmune diseases (AID) not only by producing autoantibodies but also by secreting cytokines and by presenting autoantigens. Changes in DNA methylation, histone modifications, and miRNA expression, the hallmarks of epigenetic failure, characterize B cells isolated from patients with AID, highlighting the contribution of epigenetic processes to autoreactivity. Additional evidence of epigenetic involvement in the development of B cell autoreactivity comes from in vivo and in vitro studies using DNA demethylating agents as accelerating factors or histone deacetylase inhibitors as repressing factors. As a result, a better understanding of the altered epigenetic processes in AID and in particular in B cells opens perspectives for the development of new therapeutics.

B lymphocytes from patients with systemic lupus erythematosus (SLE) are characterized by reduced expression levels of membrane CD5. Recent studies from our laboratory have revealed that the level of membrane CD5 is determined by the relative level of two alternative CD5 isoforms; CD5-E1A, which is expressed on the membrane, and CD5-E1B, which is retained in the cytoplasm. Using bisulfite sequencing and methylation-sensitive endonuclease assays we show that the promoter for the alternative CD5-E1B isoform is demethylated in B cells from patients with SLE but not in healthy controls. We go on to show that differential methylation is more pronounced following BCR engagement. As a result of this demethylation, CD5-E1B mRNA is transcribed at the expense of CD5-E1A mRNA transcription. We provide further evidence that production of high IL-6 levels by SLE B cells abrogates the ability of SLE B cells to induce DNA methyl transferase (DNMT1) and then to methylate DNA, an effect that is reversed in the presence of a blocking Ab to the IL-6 receptor. The pattern of demethylation of CpG islands in the CD5-E1B promoter in SLE B cells is similar to those in B cells from healthy controls stimulated in the presence of IL-6, or treated with the methylation inhibitor PD98059. The study reveals that engagement of the BCR with constitutive IL-6 down-regulates the level of membrane CD5, which negatively regulates BCR signaling, in SLE B cells. This altered signaling could, in turn, promote the activation and expansion of autoreactive B cells in SLE patients.

In human breast cancer spatially organized Tumor Infiltrating Lymphocytes (TILs) have been associated with effective therapeutic responses and favorable clinical outcomes.[1-3] This study investigates the potential for using InfraRed (IR) imaging to identify and characterize infiltrating lymphocytes in breast tumors, focusing on CD4+, CD8+ T lymphocytes and B lymphocytes (CD20+). Fourier Transform InfraRed (FTIR) spectroscopy coupled with microscopy is an emerging tool in cancer research and diagnosis.[4,5] IR imaging can probe the chemical composition and molecular structure of cells and tissues, thereby providing a global and unique signature for all cellular constituents. Compared with standard techniques used for identification and characterization of immune cells in tissue sections, IR imaging has numerous advantages, including no requirement for staining or automation. In this study, infrared spectra of lymphocyte subpopulations were recorded for lymphocytes in FFPE tissue sections from tonsils and breast tumors. Samples were deposited on a BaF2 window and the spectroscopic imaging data acquired in transmission mode using a Hyperion imaging system (Bruker) equipped with a focal plane array detector. The specific lymphocyte subpopulations present in each region were evaluated using hematoxyline & eosin (HE) and immunofluorescent (IF) staining of adjacent tissue sections. Statistical analyses indicate that IR spectra obtained from the CD4+, CD8+ or CD20+ lymphocyte subpopulations in tonsils are significantly different and identifiable relative to the other subpopulations. These data suggest that FTIR imaging can be used to identify specific lymphocyte subpopulations in tissues based on their spectral features. Our current work is testing this hypothesis to examine immune infiltrates in breast tumor tissue and these data will be presented at the meeting. [1] C. Gu-Trantien et al. 2013. J. Clin. Invest. 2013, 123(7) pp. 2873–2892. [2] C. Gu-Trantien and K. Willard-Gallo. Oncoimmunol. 2013, 2(10):e26066. [3] C. Denkert et al., J. Clin. Oncol. 2010, 28(1), pp. 105-113. [4] G. Bellisola and C. Sorio, Am J. Cancer Res. 2012, 2(1), pp. 1-21. [5] J. Nallala et al., Cytometry A. 2013, 83(3), pp. 294-300. Citation Format: Magali Verdonck, Soizic Garaud, Laurence Buisseret, Hugues Duvillier, Christine Desmedt, Roland de Wind, Christos Sotiriou, Karen Willard-Gallo, Erik Goormaghtigh. Characterization of tumor infiltrating lymphocytes in human breast cancer by infrared imaging. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A34.

Recent studies in breast cancer (BC) show that strong links exist between the host immune response and clinical outcome. Immune cells have been associated with both pro-tumor and anti-tumor activities with the balance between key players likely determining the ultimate clinical result. In an effort to understand how protective immune responses are generated we performed a prospective analysis of tumor infiltrating lymphocytes (TIL) in patients with primary BC. TIL present in fresh tissue homogenates (GentleMACS with no enzymatic digestion) from untreated primary breast tumors (n=120) were immunophenotyped using 10-color flow cytometry. TIL were compared for density and cellular composition to non-adjacent non-tumor breast tissue from the same patient (NANT, n=116) as well as to normal breast tissue from mammary reductions (n=28). TIL organization and distribution in tumors was analyzed using immunohistochemistry or immunofluorescence (confocal microscopy) on paraffin sections from patients in the series. Analysis of fresh tissues revealed that the density (absolute numbers of leukocytes) in normal tissue and NANT are remarkably similar (median: 4.4-vs-5.7 CD45+ cells/mm3 of tissue, respectively) and could be used to establish a cutoff for TIL negative tumors. Using NANT as an internal control, 64.5% tumors were TIL negative (minimally infiltrated). Among total leukocytes, tumors have higher frequencies of CD19+ B cells and CD4+ T cells compared with normal tissue. Furthermore, the number of CD4+ are higher than CD8+ in TIL positive tumors, which is an inversion of the CD4/CD8 ratio in TIL negative tumors (the latter ratio is similar between TIL negative tumors and NANT). In all of the breast tissues (normal, NANT and tumor) the CD4+ and CD8+ T cells are predominantly CD45RO+ memory cells, with a significant proportion of these TIL expressing the immune checkpoint receptor PD-1. In contrast, higher numbers of naïve relative to memory B cells were generally detected in both normal and tumor tissues but the balance between these B cell populations was more heterogeneous than for T cells. The distribution pattern of TILs (CD4+, CD8+ and B cells) in infiltrated tumors was both widely dispersed in some regions of the tumor bed and minimally infiltrated in other areas. Lymphocyte aggregates were principally concentrated in the stromal and peritumoral regions, with these areas being a preferential environment for B cells. Closer examination of the lymphoid aggregates revealed they were tertiary lymphoid structures containing a T cell zone predominantly composed of CD4+ T cells and a B cell follicle with an active germinal center. These data suggest that establishment of organized immune structures adjacent to the tumor bed is an effective mechanism for generating anti-tumor memory T and B cell responses. Citation Format: Laurence Buisseret, Soizic Garaud, Hugues Duvillier, Celine Naveaux, Sebastien Duquenne, Alexandre de Wind, Christos Sotiriou, Karen Willard-Gallo. A prospective study of lymphocytes infiltrating the breast cancer microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1105. doi:10.1158/1538-7445.AM2014-1105

Recent advances in tumor immunology show an important link exists between tumor infiltrating lymphocytes (TIL) and patient outcome. In human breast cancer (BC) more extensive lymphocyte infiltration is associated with a better prognosis and can also predict responses to pre-operative chemotherapy. Tertiary lymphoid structures (TLS) detected at the tumor periphery have been correlated with a good prognosis in lung and colorectal cancer. Our recent work was the first to demonstrate that TLS are present in BC, located adjacent to the tumor bed in extensively infiltrated tumors. TLS organization is similar to a lymph node, with a CD3+ T-cell zone adjacent to a CD20+ B-cell follicle where germinal centers containing CD23+ follicular dendritic cells, Bcl6+ TFH cells and Ki67+ cells are found. Our laboratory recently discovered that TLS-associated CD4+ Tfh cells are present in extensively infiltrated tumors and showed they signal a good prognosis. This important presence of Tfh-containing TLS adjacent to the tumor bed suggests that B-cells may also play a role in generating effective anti-tumor immune responses. We therefore undertook to fully characterize the B-cells infiltrating human BC. Flow cytometric analysis of fresh breast tumor homogenates detected an increase in CD45+ leukocytes in all BC subtypes compared with normal and non-tumor non-adjacent (NANT) breast tissue irrespective of lymphocyte infiltration levels. The infiltrate was dominated by CD3+ T-cells (65-86% of CD45+) in normal and malignant tissues. In contrast, B-cells were elevated in tumors compared to normal and NANT tissue, particularly in extensively infiltrated high proliferative BC subtypes. This increase was paralleled by a relative decrease in CD8+ T-cells. CD45/CD19/CD38/IgD labeling revealed that approximately 50% of the infiltrating B-cells are memory cells in contrast to normal tissues, which contain less than 15%. Centroblasts and centrocytes, which are follicular B cells, were significantly increased in extensively infiltrated tumors in association with Tfh cells. We assessed immunoglobulin isotypes in primary supernatants from breast tissue homogenates, detecting decreased IgA and increased IgG and IgM per mm3 in the tumor. Patient sera, particularly from high proliferative subtypes, had elevated IgG4 compared with low proliferative subtypes and healthy donors. Immunofluorescent analysis of embedded tissues show that TLS contain a marginal zone region(CD20+CD27+IgD-), a follicular mantle zone (CD20+IgD+) and a germinal center zone (CD20+Ki67+CD35+CD21+PD-1+). Tumor-associated TLS are surrounded by T-cells (CD4+ and CD8+). These data reveal that when B-cells are present in breast tumors they are principally associated with organized TLS and may therefore play an important functional role in comprehensive anti-tumor immune responses, an aspect currently under further investigation. Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu, Edoardo Migliori, Jean-Nicolas Lodewyckx, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Karen Willard-Gallo. Characterization of B-cells infiltrating human breast cancer and their presence in peritumoral tertiary lymphoid structures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1669. doi:10.1158/1538-7445.AM2014-1669

Molecular approaches such as gene expression profiling have improved the classification of human breast cancer (BC) by identifying molecular subtypes and demonstrating that the immune infiltrate is an important prognostic or predictive factor. Our prospective study of freshly isolated CD4+ T cells infiltrating (TIL) BC discovered that along with Th1, Th2 and Th17 effector memory and Treg subpopulations the newest CD4+ Th subset, T follicular helper (Tfh) cells, was also present in the tumor. Comparison of extensive versus minimally-infiltrated tumors led to our finding that extensive immune infiltrates are distinguished by Tfh cells located in tertiary lymphoid structures (TLS) next to the tumor bed. TLS are highly organized with a CD3+ T cell zone adjacent to a CD20+ B cell follicle with germinal centers containing Bcl6+ Tfh cells, CD23+ follicular dendritic cells and Ki67+ cells. Tfh cells are known to play a critical role in generating antibody producing plasma cells and memory B cells. We demonstrated that Tfh cells are the principal cellular source of the B cell chemoattractant CXCL13 in BC. Thus, a Tfh presence paralleled the TLS incidence and together they were associated with an increased peritumoral B cell presence and a higher frequency of CD8+ T cells in the tumor bed. Our recent work shows that approximately 50% of the infiltrating B cells are memory cells in contrast to normal and non-tumor non-adjacent breast tissue (controls, less than 15%). A significant B cell presence is found in extensively infiltrated high proliferative BC subtypes with germinal center centroblasts and centrocytes consistently associated with a Tfh cell presence. We assessed immunoglobulin isotypes in supernatants of fresh breast tissue homogenates, detecting decreased IgA and increased IgG and IgM in the tumor tissue compared to controls. Patient sera, particularly from patients with high proliferative BC subtypes, had elevated IgG3 and IgG4 compared to low proliferative tumors and healthy donors. Immunofluorescent analysis found the majority of B cells infiltrating tumors have germinal center characteristics (IgD+Ki67+CD35+CD21+PD-1+) and are clustered with Tfh in the B cell follicles surrounded by a T cell zone, which together create the tumor-associated TLS. Based on their specificity for TLS, we derived a scoring system, referred to as immunological grade, to measure the extent of the lymphocytic infiltrate and the level of immune organization. This system is based on a morphological score (the density of the lymphocyte infiltrate determined by CD3/CD20 immunohistochemistry plus the number and size of peritumoral TLS assessed by two pathologists) together with a molecular score [an 8 gene qRT-PCR Tfh signature that predicts long term survival in an untreated patient cohort with >10-year survival (n = 794) or pathological complete response in patients treated with preoperative chemotherapy (n = 996)]. We are testing this immunological grading system on a large retrospective series of patients to determine its value in evaluating a patient's anti-tumor immune response in pre-operative biopsies and/or the primary tumor at surgery as a new marker for use in treatment decisions. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-02.