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

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

Introduction: Tumor banks make a considerable contribution to translational research. Using emerging molecular tests on frozen material facilitates the development of new diagnostic and therapeutic strategies, especially in rare cases. However, standard quality control schemes are lacking in the current literature. Methods: In 2017, we have conducted a robust quality control test on 100 of 15,000 fresh frozen samples collected between 2000 and 2013 at the Jules Bordet Tumor Bank (Brussels). RNA and DNA extraction was done. The quality of RNA, DNA and proteins were evaluated, respectively by measuring RNA Integrity Number (RIN), by checking Electrophoretic Integrity (EI) and by performing Immunohistochemistry staining (IHC). A score, ranging from poor (1) to excellent (4), was attributed based on technical analysis. Results: RNA purity was scored 4 in 97% of the cases, 3 in 2%, and 2 in 1%. RIN scores were similarly 4 in 89%, 3 in 10%, and 2 in 1% of the cases. DNA purity was scored 4 in 94% and 3 in 6%, EI was scored 4 in 100% of the cases. Despite morphology loss after freezing, HER2, ER, and Ki67 IHC stainings yielded a score of 4 in the majority of samples. Furthermore, participating in the ISBER Proficiency Testing helped us validate our techniques and the technician's work. Seven processing schemes were carried out, the scores obtained were very satisfactory (20/27) or satisfactory (7/27). Conclusion: Tumor Banks can be precious for translational research. Nevertheless, firm quality controls should be applied to ensure high quality material delivery. Only then can biobanks contribute to diagnostics, biomarkers discovery and reliable molecular test development., SCOPUS: ar.j, info:eu-repo/semantics/published

Tumor-infiltrating B cells (TIL-B) in breast cancer (BC) have previously been associated with improved clinical outcomes; however, their roles in tumor immunity are not currently well known. This study confirms and extends the correlation between higher TIL-B densities and positive outcomes through an analysis of HER2+ and triple-negative BC patients from the BIG 02-98 clinical trial (10-year median follow-up). Fresh tissue analyses identify an increase in TIL-B density in untreated primary BC compared with normal breast tissues, which is associated with global, CD4+, and CD8+ tumor infiltrating lymphocytes (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 T follicular helper (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 that TIL-B are responsive to B cell receptor (BCR) stimulation ex vivo, express activation markers, and produce cytokines and Igs 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 promote effective antitumor immunity at the tumor site., SCOPUS: ar.j, info:eu-repo/semantics/published

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

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma (<5% of Hodgkin’s lymphomas) predominantly affecting the middle-aged man, with an indolent behavior. Given the rare occurrence of this lymphoma, there are currently no clear guidelines for initial treatment or relapse. In this report, we present the follow-up of 2 patients treated by radioimmunotherapy for first relapse of their NLPHL. Both patients were initially treated with rituximab and relapsed 1 year after the end of their treatment., 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-1hiICOSintFOXP3hi 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., SCOPUS: ar.j, info:eu-repo/semantics/published

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

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

Confocal laser endomicroscopy (CLE) enables in-vivo, real-time, imaging of tissues with a micron-scale resolution through a fiber optic probe. CLE could be a valuable tool for the detection and characterization of suspicious (dysplastic) areas on the uterine cervix in a minimally invasive manner. This study evaluates the technical feasibility and safety of CLE on the cervix. The study also aims to create a preliminary iconography of normal and dysplastic squamous and columnar cervical epithelium. In-vivo CLE was performed on nine patients scheduled for a cervical loop electric excision procedure for high-grade superficial intraepithelial lesions. The CLE images were compared with standard hematoxylin and eosin analysis of loop electric excision procedure specimens. The histopathological diagnosis on the surgical specimen was established as per standard of care. CLE images were then reviewed by pathologists to point out specific histopathological features. pCLE of the exocervix and the transformation zone was performed successfully on seven out of nine patients. Uninterpretable images were obtained in two other cases: one using the AlveoFlex and one using the GastroFlex UHD after the application of acetic acid 2%. A total of 82.5% of the sequences recorded with the GastroFlex were suitable for interpretation. No adverse event or complications occurred. CLE enables proper in-vivo imaging of healthy and dysplastic cervical tissue. Images correlate well with the histopathological features established through traditional histology. Future blinded prospective analysis will determine the reliability of the realtime diagnosis and its potential use in the assessment and treatment of cervical lesions., SCOPUS: ar.j, info:eu-repo/semantics/published