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

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

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

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

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma (

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

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

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.

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.

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.

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.

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

The presence of tumor-infiltrating lymphocytes (TIL), reflecting host immune activity, is frequently correlated with better clinical outcomes, particularly in HER2-positive and triple-negative breast cancer. Recent findings suggest that organization of immune infiltrates in tertiary lymphoid structures also has a beneficial effect on survival. This study investigated inter- and intra-observer variation in TIL assessment using conventional hematoxylin-eosin versus immunohistochemical staining to identify immune cells. Global, intratumoral, and stromal TIL, as well as tertiary lymphoid structures were scored independently by experienced pathologists on full-face tumor sections (n=124). The fidelity of scoring infiltrates in core biopsies compared to surgical specimens, and pathological assessment compared to quantitative digital analysis was also evaluated. The inter-observer concordance correlation coefficient was 0.80 for global, 0.72 for intratumoral, and 0.71 for stromal TIL, while the intra-observer concordance correlation coefficient was 0.90 for global, 0.77 for intratumoral, and 0.89 for stromal TIL using immunohistochemical stains. Correlations were lower with hematoxylin-eosin stains, particularly for intratumoral TIL, while global scores had the highest concordance correlation coefficients. Our study concluded that tertiary lymphoid structures are accurately and consistently scored using immunohistochemical but not hematoxylin-eosin stains. A strong association was observed between TIL in core biopsies and surgical samples (R2=0.74) but this did not extend to tertiary lymphoid structures (R2=0.26). TIL scored by pathologists and digital analysis were correlated but our analysis reveals a constant bias between these methods. These data challenge current criteria for TIL and tertiary lymphoid structure assessment in breast cancer and recommend that how pathologists evaluate immune infiltrates be reexamined for future studies.

In this era of Highly Active Antiretroviral Therapy (HAART), we see that incidence of neoplasic diseases among patients affected by Human Immonudeficiency Virus (HIV) is increasing. Plasmablastic lymphoma (PBL) is an example of neoplastic condition specific to HIV patients. It is a subtype of diffuse large B-cell lymphoma (DLBCL) recently described in the 2008 WHO classification of lymphoproliferative disorder. PBL is characterized by its aggressive nature and plasmacytic differentiation and usually occurs in the oral cavity or jaw of HIV-infected individuals. However, several cases of PBL involving extraoral sites have been reported in immunocompetent individuals. This entity is a diagnostic challenge and need integration of clinical, pathological, genetic and microbiologic features. Several types of lymphoma present with plasmablastoid differenciation and may give rise to some difficulties for a differential diagnosis. We give some anatomopathological, molecular and genetic details for each subtype and we will illustrate the difficulty for the diagnosis with two cases. The first case is a patient with plasmablastic lymphoma that present with unusual negative immunoreactivity for CD 138 (10% of PBL cases). The second case has clinical, epidemiological and pathological characteristic of plasmablastic lymphoma but HHV8 positivity is a criteria for considering extra-cavitary PEL as the most relevant diagnosis.

Intraarterial administration of (90)Y microspheres to the spleen in patients with malignant lymphoma was mentioned once in the literature in 1973. This case study illustrates the potential indication of selective internal radiotherapy in a heavily pretreated patient with highly refractory disease with a marginal zone lymphoma in leukemic phase and symptomatic splenomegaly. We describe the clinical course of disease; the biological and clinical response to the treatment after radioembolization; and simulation and dosimetry by multimodal imaging via single-photon emission computed tomography and computed tomography. The advantages of radioembolization for the management of lymphomatous splenomegaly are discussed.

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.

Retiform haemangioendothelioma is a locally aggressive, very rarely metastasizing vascular lesion. Histologically, it is characterized by distinctive arborizing blood vessels resembling "rete testis" and lined by endothelial cells with characteristic hobnail morphology. We present an additional case, in the leg of a 64-year-old patient. We discuss the classification of hemangioendotheliomas. The term hemangioendothelioma should be restricted to vascular tumours of "intermediate malignancy" but has been used to designate tumours with variable histological features and clinical behaviour. Spindle cell hemangio(endothelio)ma is currently regarded as a benign reactive lesion. Kaposiform hemangioendothelioma is potentially lethal due to consumption coagulopathy but no metastasizing case has been reported. Epithelioid hemangioendothelioma is associated with a significant metastatic risk and has been included in the category of malignant vascular tumors. The vascular lesions fulfilling the strict definition of hemangioendothelioma include retiform hemangioendothelioma, papillary intralymphatic angioendothelioma "Dabska's tumor", composite hemangioendothelioma and perhaps the controversial polymorphic hemangioendothelioma.

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers., SCOPUS: ar.j, FLWOA, info:eu-repo/semantics/published

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.

Retiform haemangioendothelioma is a locally aggressive, very rarely metastasizing vascular lesion. Histologically, it is characterized by distinctive arborizing blood vessels resembling "rete testis" and lined by endothelial cells with characteristic hobnail morphology. We present an additional case, in the leg of a 64-year-old patient. We discuss the classification of hemangioendotheliomas. The term hemangioendothelioma should be restricted to vascular tumours of "intermediate malignancy" but has been used to designate tumours with variable histological features and clinical behaviour. Spindle cell hemangio(endothelio)ma is currently regarded as a benign reactive lesion. Kaposiform hemangioendothelioma is potentially lethal due to consumption coagulopathy but no metastasizing case has been reported. Epithelioid hemangioendothelioma is associated with a significant metastatic risk and has been included in the category of malignant vascular tumors. The vascular lesions fulfilling the strict definition of hemangioendothelioma include retiform hemangioendothelioma, papillary intralymphatic angioendothelioma "Dabska's tumor", composite hemangioendothelioma and perhaps the controversial polymorphic hemangioendothelioma.

Background: Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histo-pathological parameters.Material and methods: The characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER and HER2 status. Mutations were classified as ‘oncogenic’ in case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep re-sequencing using an orthogonal platform. Whole genome rearrangement screen was further conducted in 8/36 patients.Results: Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3 and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than the homogeneous ones. Genome-wide analysis of a limited number of MFBC nevertheless identified a common somatic background in all studied MFBC, including those with no mutation in common between the lesions. Conclusion and perspectives: As the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings, based on the targeted sequencing of cancer genes in 36 MFBC tumors, highlight the presence of genomic inter-lesion heterogeneity in one-third of the cases despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers. [CD, DF, and EP contributed equally to this work. PJC and CS contributed equally to this work.] Citation Format: Christine Desmedt, Debora Fumagalli, Elisabetta Pietri, Gabriele Zoppoli, Serena Nik-Zainal, Gunes Gundem, David Brown, Francois Rothe, Samira Majjaj, Anna Garuti, Enrico Carminati, Sherene Loi, Thomas Van Brussel, Marion Maetens, Laura Mudie, Delphine Vincent, Naima Kheddoumi, Luigi Serra, Ilaria Massa, Alberto Ballestrero, Dino Amadori, Roberto Salgado, Alexandre de Wind, Diether Lambrechts, Martine Piccart, Denis Larsimont, Peter J. Campbell, Christos Sotiriou. Uncovering the genomic heterogeneity of multifocal breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT135. doi:10.1158/1538-7445.AM2015-CT135

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

CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. The goal of this project was to characterize CD4+ T lymphocytes infiltrating primary human breast tumors (TIL), analyze T cell signaling pathways influenced by the tumor microenvironment, correlate differential gene expression with the presence of a specific CD4+ T cell subset(s) and identify a profile predictive of positive patient outcome. Affymetrix U133 Plus 2.0 arrays were used to analyze CD4+ T cells freshly isolated from the primary tumor, axillary lymph node (LN) and blood (PB) of patients with ER+ and ER- invasive breast carcinoma in the discovery set (10 patients). Microarrays, flow cytometry and/or qRT-PCR were used to extend and confirm the data in CD4+ TIL isolated from >60 primary breast tumors as well as purified CD4+ T cells from healthy donors PB treated with supernatant (SN) derived from fresh breast tumor tissue. Assessment of the relative distribution of infiltrating mononuclear cells revealed that the CD4+ T cell subset was consistently the principal component. We detected gene expression changes in the CD4+ TIL compared to patient's LN and PB counterparts that reflected altered cellular signaling pathways, including significant suppression of the TCR/CD3 complex and downstream signaling molecules along with a highly restricted pattern of Th cytokine and chemokine expression. A comparison with publically available Th profiling microarray data revealed that the TIL have characteristics of an activated memory effector CD4+ T cell phenotype, with all the major subsets (Th1, Th2, Th17, Treg, Tfh) represented. Although very few differences were detected in a direct comparison between CD4+ TIL from ER+ and ER- tumors, important changes in TIL (including a Th subset skew and differential cytokine expression) from extensive versus minimally infiltrated tumors were observed, with those containing an extensive CD4+ T cell infiltrate associated with a better clinical outcome. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight. These experiments revealed that the rested TIL reverse expression in a number of critical cytokine and signaling genes. PB CD4+ T cells from healthy donors treated with primary tumor SN also displayed significant changes in gene expression, gaining >75% similarity to freshly isolated CD4+ TIL. These experiments further found that activation of CD4+ T cells in conjunction with tumor SN treatment rapidly suppressed stimulation-induced changes in gene expression. Together our data suggest that although activated CD4+ T cells infiltrating breast tumors are quickly suppressed, tumors that recruit higher levels of CD4+ T cells, even in the face of rapid immunosuppression, produce a better memory T cell pool that is ultimately capable of maintaining effective anti-tumor immunity in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3541. doi:1538-7445.AM2012-3541