Deciphering the maturation of tertiary lymphoid structures in cancer and inflammatory diseases of the digestive tract using imaging mass cytometry.

Persistent inflammation can promote the development of tertiary lymphoid structures (TLS) within tissues resembling secondary lymphoid organs (SLO) such as lymph nodes (LN). The composition of TLS across different organs and diseases could be of pathophysiological and medical interest. In this work, we compared TLS to SLO in cancers of the digestive tract and in inflammatory bowel diseases. Colorectal and gastric tissues with different inflammatory diseases and cancers from the department of pathology of CHU Brest were analyzed based on 39 markers using imaging mass cytometry (IMC). Unsupervised and supervised clustering analyses of IMC images were used to compare SLO and TLS. Unsupervised analyses tended to group TLS per patient but not per disease. Supervised analyses of IMC images revealed that LN had a more organized structure than TLS and non-encapsulated SLO Peyer's patches. TLS followed a maturation spectrum with close correlations between germinal center (GC) markers' evolution. The correlations between organizational and functional markers made relevant the previously proposed TLS division into three stages: lymphoid-aggregates (LA) (CD20+CD21-CD23-) had neither organization nor GC functionality, non-GC TLS (CD20+CD21+CD23-) were organized but lacked GC's functionality and GC-like TLS (CD20+CD21+CD23+) had GC's organization and functionality. This architectural and functional maturation grading of TLS pointed to differences across diseases. TLS architectural and functional maturation grading is accessible with few markers allowing future diagnostic, prognostic, and predictive studies on the value of TLS grading, quantification and location within pathological tissues in cancers and inflammatory diseases.

Immune-checkpoint inhibitor use in patients with cancer and pre-existing autoimmune diseases.

Immune-checkpoint inhibitors (ICIs) have dramatically changed the management of advanced cancers. Designed to enhance the antitumour immune response, they can also cause off-target immune-related adverse events (irAEs), which are sometimes severe. Although the efficacy of ICIs suggests that they could have wide-ranging benefits, clinical trials of the drugs have so far excluded patients with pre-existing autoimmune disease. However, evidence is accumulating with regard to the use of ICIs in this 'at-risk' population, with retrospective data suggesting that they have an acceptable safety profile, but that there is a risk of disease flare or other irAE occurrence. The management of immunosuppressive drugs at ICI initiation in patients with autoimmune disease (or later in instances of disease flare or irAE) remains a question of particular interest in clinical practice, in which there is always a search for the balance between protecting against autoimmunity and ensuring a good tumour response. Although temporary use of immunosuppressants seems safe, prolonged use or use at ICI initiation might hamper the antitumour immune response, prompting clinicians to use the minimal efficient immunosuppressive regimen. However, a new paradigm is emerging, in which inhibitors of TNF or IL-6 could have synergistic effects with ICIs on tumour response, while also preventing severe irAEs. If confirmed, this 'decoupling' effect on toxicity and efficacy could change therapeutic practice in this field. Knowledge of the current use of ICIs in patients with pre-existing autoimmune disease, particularly with regard to the use of immunosuppressive drugs and/or biologic DMARDs, can help to guide clinical practice.

A Rare Case of Hepatic Vanishing Bile Duct Syndrome Occurring after Combination Therapy with Nivolumab and Cabozantinib in a Patient with Renal Carcinoma.

Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) significantly improve the outcomes of patients with advanced clear cell renal cell carcinoma (ccRCC); however, high-grade toxicities can occur, particularly during combination therapy. Herein, we report a patient with advanced metastatic ccRCC, who developed grade 4 cholestasis during combined therapy with nivolumab and cabozantinib. After the exclusion of common disorders associated with cholestasis and a failure of corticosteroids (CS), a liver biopsy was performed that demonstrated severe ductopenia. Consequently, a diagnosis of vanishing bile duct syndrome related to TKI and ICI administration was made, resulting in CS discontinuation and ursodeoxycholic acid administration. After a 7-month follow-up, liver tests had returned to normal values. Immunological studies revealed that our patient had developed robust T-cells and macrophages infiltrates in his lung metastasis, as well as in skin and liver tissues at the onset of toxicities. At the same time, peripheral blood immunophenotyping revealed significant changes in T-cell subsets, suggesting their potential role in the pathophysiology of the disease.

Low-Dose Nivolumab with or without Ipilimumab as Adjuvant Therapy Following the Resection of Melanoma Metastases: A Sequential Dual Cohort Phase II Clinical Trial.

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.

Fluorescent Multiplex Immunohistochemistry Coupled With Other State-Of-The-Art Techniques to Systematically Characterize the Tumor Immune Microenvironment.

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.

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity.

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-1hiICOSint 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-ß-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.

Persistent anti-tumor response in cancer patients experiencing pneumonitis related to immune checkpoint blockade.

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

Downregulation of the FTO m6A RNA demethylase promotes EMT-mediated progression of epithelial tumors and sensitivity to Wnt inhibitors.

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 N6-methyladenosine (m6A), 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 m6A 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.

Inhibition of RANK signaling in breast cancer induces an anti-tumor immune response orchestrated by CD8+ T cells.

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.

Retrospective analysis of the immunogenic effects of intra-arterial locoregional therapies in hepatocellular carcinoma: a rationale for combining selective internal radiation therapy (SIRT) and immunotherapy.

BACKGROUND: Immunotherapy represents a promising option for treatment of hepatocellular carcinoma (HCC) in cirrhotic patients but its efficacy is currently inconsistent and unpredictable. Locoregional therapies inducing immunogenic cell death, such as transarterial chemoembolization (TACE) or selective internal radiation therapy (SIRT), have the potential to act synergistically with immunotherapy. For the development of new approaches combining locoregional treatments with immunotherapy, a better understanding of the respective effects of TACE and SIRT on recruitment and activation of immune cells in HCC is needed. To address this question, we compared intra-tumor immune infiltrates in resected HCC after preoperative treatment with TACE or SIRT. METHODS: Data fromr patients undergoing partial hepatectomy for HCC, without preoperative treatment (SURG, n = 32), after preoperative TACE (TACE, n = 16), or preoperative SIRT (n = 12) were analyzed. Clinicopathological factors, tumor-infiltrating lymphocytes (TILs), CD4+ and CD8+ T cells, and granzyme B (GZB) expression in resected HCC, and postoperative overall and progression-free survival were compared between the three groups. RESULTS: Clinicopathological and surgical characteristics were similar in the three groups. A significant increase in TILs, CD4+ and CD8+ T cells, and GZB expression was observed in resected HCC in SIRT as compared to TACE and SURG groups. No difference in immune infiltrates was observed between TACE and SURG patients. Within the SIRT group, the dose of irradiation affected the type of immune infiltrate. A significantly higher ratio of CD3+ cells was observed in the peri-tumoral area in patients receiving < 100 Gy, whereas a higher ratio of intra-tumoral CD4+ cells was observed in patients receiving > 100 Gy. Postoperative outcomes were similar in all groups. Irrespective of the preoperative treatment, the type and extent of immune infiltrates did not influence postoperative survival. CONCLUSIONS: SIRT significantly promotes recruitment/activation of intra-tumor effector-type immune cells compared to TACE or no preoperative treatment. These results suggest that SIRT is a better candidate than TACE to be combined with immunotherapy for treatment of HCC. Evaluation of the optimal doses for SIRT for producing an immunogenic effect and the type of immunotherapy to be used require further evaluation in prospective studies.

Tumor-Derived Thymic Stromal Lymphopoietin Expands Bone Marrow B-cell Precursors in Circulation to Support Metastasis.

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.

Tumor infiltrating B-cells signal functional humoral immune responses in breast cancer.

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.

BRCA gene mutations do not shape the extent and organization of tumor infiltrating lymphocytes in triple negative breast cancer.

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.

Age-related changes in the BACH2 and PRDM1 genes in lymphocytes from healthy donors and chronic lymphocytic leukemia patients.

BACKGROUND: 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. METHODS: Lymphocyte subsets from 60 healthy donors, aged from 20 to 90 years, and 41 untreated chronic lymphocytic leukemia patients were studied. BACH2 and PRDM1 gene expression was analyzed by real-time quantitative PCR. BACH2 gene expression was correlated with its protein expression. Lymphocyte apoptosis was evaluated after intracellular oxidative stress-inducing etoposide treatment of T and B cells. RESULTS: Our analysis shows BACH2 mRNA downregulation with age in healthy donor CD4+, CD8+ T-cells and CD19+ B-cells. Decreased BACH2 expression was also correlated with an age-related reduction in CD8 + CD28+ T-cells. We found a strong correlation between age-related BACH2 downregulation and decreased CD4+ T-cell and CD19+ B-cell apoptosis. PRDM1, as expected, was significantly upregulated in CD4+ T-cells, CD8+ T-cells and CD19+ B-cells, and inversely correlated with BACH2. A comparison of untreated chronic lymphocytic leukemia patients with age-matched healthy donors reveals that BACH2 mRNA expression was further reduced in CD4+ T-cells, CD8+ T-cells and leukemic-B cells. PRDM1 gene expression was consequently significantly upregulated in CD4+ and CD8+ T-cells in chronic lymphocytic leukemia patients but not in their leukemic B-cells. CONCLUSION: Overall, our data suggest that BACH2 and PRDM1 genes are significantly correlated with age in human immune cells and may be involved in immunosenescence.

FOXP1 negatively regulates tumor infiltrating lymphocyte migration in human breast cancer.

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 TGFß 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.

Antigen Specificity and Clinical Significance of IgG and IgA Autoantibodies Produced in situ by Tumor-Infiltrating B Cells in Breast Cancer.

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.

Quantifying Tertiary Lymphoid Structure-Associated Genes in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissues.

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.

Immunity drives TET1 regulation in cancer through NF-κB.

Ten-eleven translocation enzymes (TET1, TET2, and TET3), which induce DNA demethylation and gene regulation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are often down-regulated in cancer. We uncover, in basal-like breast cancer (BLBC), genome-wide 5hmC changes related to TET1 regulation. We further demonstrate that TET1 repression is associated with high expression of immune markers and high infiltration by immune cells. We identify in BLBC tissues an anticorrelation between TET1 expression and the major immunoregulator family nuclear factor κB (NF-κB). In vitro and in mice, TET1 is down-regulated in breast cancer cells upon NF-κB activation through binding of p65 to its consensus sequence in the TET1 promoter. We lastly show that these findings extend to other cancer types, including melanoma, lung, and thyroid cancers. Together, our data suggest a novel mode of regulation for TET1 in cancer and highlight a new paradigm in which the immune system can influence cancer cell epigenetics.

Immune Infiltration in Invasive Lobular Breast Cancer.

Background: Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer after invasive ductal cancer (IDC). Here, we aimed at evaluating the prevalence, levels, and composition of tumor-infiltrating lymphocytes (TILs) and their association with clinico-pathological and outcome variables in ILC, and to compare them with IDC. Methods: We considered two patient series with TIL data: a multicentric retrospective series (n = 614) and the BIG 02-98 study (n = 149 ILC and 807 IDC). We compared immune subsets identified by immuno-histochemistry in the ILC (n = 159) and IDC (n = 468) patients from the Nottingham series, as well as the CIBERSORT immune profiling of the ILC (n = 98) and IDC (n = 388) METABRIC and The Cancer Genome Atlas patients. All ILC/IDC comparisons were done in estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative tumors. All statistical tests were two-sided. Results: TIL levels were statistically significantly lower in ILC compared with IDC (fold-change = 0.79, 95% confidence interval = 0.70 to 0.88, P < .001). In ILC, high TIL levels were associated with young age, lymph node involvement, and high proliferative tumors. In the univariate analysis, high TIL levels were associated with worse prognosis in the retrospective and BIG 02-98 lobular series, although they did not reach statistical significance in the latter. The Nottingham series revealed that the levels of intratumoral but not total CD8+ were statistically significantly lower in ILC compared with IDC. Comparison of the CIBERSORT profiles highlighted statistically significant differences in terms of immune composition. Conclusions: This study shows differences between the immune infiltrates of ER-positive/HER2-negative ILC and IDC in terms of prevalence, levels, localization, composition, and clinical associations.