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supplementary Table S1 to S3; Supplementary Figure S1 to S9

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS–STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.

Disease free survival (DFS) and overall survival (OS) analysis according to AR mRNA expression levels in BC subtypes

Data sets for gene-expression analysis with patients' and tumours' characteristics from the neoadjuvant studies

Legends to Supplementary Figures

Supplementary Tables and Figures legend

Fig.S1: CD73 prognosis in untreated breast cancer; Fig. S2: CD73 prognosis in METABRIC; Fig. S3: Multivariate analysis; Fig. S4: CD73 expression in H2N100 and TUBO; Fig. S5: H2N100 and TUBO survival data; Fig. S6: Combined treatment in NRG mice; Fig. S7: CD73 and EMT correlations

PRISMA flow chart for studies involved in clinical meta-analysis Study flow chart for gene expression pooled analysis

Purpose: Androgen receptor (AR) expression has been observed in about 70% of patients with breast cancer, but its prognostic role remains uncertain.Experimental Design: To assess the prognostic role of AR expression in early-stage breast cancer, we performed a meta-analysis of studies that evaluated the impact of AR at the protein and gene expression level on disease-free survival (DFS) and/or overall survival (OS). Eligible studies were identified by systematic review of electronic databases using the MeSH-terms “breast neoplasm” and “androgen receptor” and were selected after a qualitative assessment based on the REMARK criteria. A pooled gene expression analysis of 35 publicly available microarray data sets was also performed from patients with early-stage breast cancer with available gene expression and clinical outcome data.Results: Twenty-two of 33 eligible studies for the clinical meta-analysis, including 10,004 patients, were considered as evaluable for the current study after the qualitative assessment. AR positivity defined by IHC was associated with improved DFS in all patients with breast cancer [multivariate (M) analysis, HR 0.46; 95% confidence interval (CI) 0.37–0.58, P < 0.001] and better OS [M-HR 0.53; 95% CI, 0.38–0.73, P < 0.001]. Thirty-five datasets including 7,220 patients were eligible for the pooled gene expression analysis. High AR mRNA levels were found to confer positive prognosis overall in terms of DFS (HR 0.82; 95% CI 0.72–0.92;P = 0.0007) and OS (HR 0.84; 95% CI, 0.75–0.94; P = 0.02) only in univariate analysis.Conclusions: Our analysis, conducted among more than 17,000 women with early-stage breast cancer included in clinical and gene expression analysis, demonstrates that AR positivity is associated with favorable clinical outcome. Clin Cancer Res; 23(11); 2702–12. ©2016 AACR.

Correlation of clinical variables and AR expression in different BC subtypes: A-in Luminal A; B-in Luminal B; C-in HER2 enriched; D-in Basal like.

Association of AR mRNA levels and individual genes and gene signatures (GS) in all BC patients

Data sets for gene-expression analysis with patients' and tumours' characteristics

Main characteristics of evaluable clinical studies included in meta-analysis for disease free survival (DFS) and overall survival (OS)

REMARK scores of eligible studies for clinical meta-analysis

Characterizing T cell populations and understanding their developmental relationships may help design more effective cancer immunotherapies. We coupled single-cell transcriptomics and T cell receptor (TCR) αβ profiling of intratumoral and peripheral T cells in ovarian cancer patients to identify transcriptional programs and infer their relationship by trajectory and TCR overlap analyses. We proposed a model of differentiation pathway from an intermediate GZMH-expressing CD8 T cell subset found in the blood and tumor that progressively reinforces the exhaustion and tissue residency programs from aCCL4-expressing cluster towardsXCL1- andCXCL13-expressing terminally exhausted cells. Inferred cell communication analysis suggests that interaction withCXCL13-expressing CD4 T cells, which we refer to as Tfh-like cells, sustains the effector function of this intermediate GZMH-expressing CD8 T cell subset. Moreover, our results suggest that Tfh-like cells attract cells expressingGPR183through the production of its ligand 7α,25 dihydroxycholesterol (7α,25-HC). Finally, we demonstrated thatGPR183is highly expressed in a subset of pre-effectorGZMK-expressing CD8 T cells and plasmacytoid dendritic cells. Collectively, our results suggest that Tfh-like cells expressing IL-21 help promote antitumor immunity against ovarian tumors by coordinating the action of immune cells responsive to 7α,25-HC.

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS–STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.

The ecto-nucleotidase CD73 is an important immune checkpoint in tumor immunity that cooperates with CD39 to hydrolyze pro-inflammatory extracellular ATP into immunosuppressive adenosine. While the role of CD73 in immune evasion of solid cancers is well established, its role in leukemia remains unclear. To investigate the role of CD73 in the pathogenesis of chronic lymphocytic leukemia (CLL), Eµ-TCL1 transgenic mice that spontaneously develop CLL were crossed with CD73−/− mice. Disease progression in peripheral blood and spleen, and CLL markers were evaluated by flow cytometry and survival was compared to CD73-proficient Eµ-TCL1 transgenic mice. We observed that CD73 deficiency significantly delayed CLL progression and prolonged survival in Eµ-TCL1 transgenic mice, and was associated with increased accumulation of IFN-γ+ T cells and effector-memory CD8+ T cells. Neutralizing IFN-γ abrogated the survival advantage of CD73-deficient Eµ-TCL1 mice. Intriguingly, the beneficial effects of CD73 deletion were restricted to male mice. In females, CD73 deficiency was uniquely associated with the upregulation of CD39 in normal lymphocytes and sustained high PD-L1 expression on CLL cells. In vitro studies revealed that adenosine signaling via the A2a receptor enhanced PD-L1 expression on Eµ-TCL1-derived CLL cells, and a genomic analysis of human CLL samples found that PD-L1 correlated with adenosine signaling. Our study, thus, identified CD73 as a pro-leukemic immune checkpoint in CLL and uncovered a previously unknown sex bias for the CD73-adenosine pathway.

Cancer treatments as cyclophosphamide and its active metabolites are highly gonadotoxic leading to follicle apoptosis and depletion. Considering the risk of subsequent infertility, fertility preservation is recommended. Beside the germ cells and gametes cryopreservation options, ovarian pharmacological protection during treatment appears to be very attractive. Meanwhile, the advances in the field of oncology have brought microRNAs into spotlight as a potential feature of cancer treatment. Herein, we investigated miRNAs expressions in response to chemotherapy using postnatal-day-3 (PND3) mouse ovaries. Our results revealed that several miRNAs are differently expressed during chemotherapy exposure. Amongst them, let-7a was the most profoundly downregulated and targets genes involved in crucial cellular processes including apoptosis. Thus we developed a liposome-based system to deliver the let-7a mimic in whole PND3 ovaries in vitro. We showed that let-7a mimic prevented the upregulation of genes involved in cell death and reduced the chemotherapy-induced ovarian apoptosis, suggesting that it can be an interesting target to preserve ovarian function. However, its impact on subsequent follicular development has to be further elucidated in vivo using an appropriate delivery system. In this study, we demonstrated that miRNA replacement approaches can be a useful tool to reduce chemotherapy-induced ovarian damage in the future.

BACKGROUND: Adjuvant therapy improves the prognosis of stage II & III colon cancer patients. Unfortunately, most patients do not benefit from this treatment. PePITA (NCT00994864) is a prospective, multicenter, non-randomized study whose primary objective is to predict the outcome of adjuvant therapy in colon cancer. METHODS: The primary objective was to determine the prognostic and predictive value of circulating tumor cell (CTC) detection before therapy and after one course of preoperative FOLFOX. RESULTS: Out of the 58 first patients accrued in PePiTA trial, 36 patients participated in the CTC companion study, of whom 32 had at least one evaluable sample. Only 5 patients (14, 95% CI = 5-30%) had ≥1 CTC/22.5 ml blood in at least one of the two timepoints with 2 patients having ≥1 CTC/22.5 ml at baseline (6, 95% CI: 1-19%). The detection rate of patients with CTCs at baseline being lower than expected, the inclusion of patients in the PePiTA CTC substudy was stopped. The limited sample size did not allow us to investigate the prognostic and predictive value of CTCs in locally advanced colon cancer. CONCLUSIONS: Our data illustrate the need for further standardized studies in order to find the most reliable prognostic/predictive biomarker in early-stage colon cancer. TRIAL REGISTRATION: This trial was prospectively registered at Jules Bordet institute ( NCT00994864 ) on the October 14, 2009. ispartof: BMC CANCER vol:19 issue:1 ispartof: location:England status: published

Introduction Mucinous carcinoma, a special histological subtype of breast cancer (BC) which accounts for ˜2% of all invasive breast tumors, is characterized by the presence of extracellular mucin, typically expresses the estrogen receptor (ER), and lacks HER2 amplification. The majority of patients with mucinous BC are older at diagnosis, rarely present axillary lymph node metastases, and are associated with a better prognosis as compared to invasive ductal cancer patients (IDC, formally referred to as “breast cancer of no special type”). So far, it is unknown what is driving the mucinous phenotype of these tumors. Here we interrogated the genomics, transcriptomics, immune infiltration and epigenetics profiles of these tumors, using a retrospective institutional series and a publicly available dataset. Patients and methods After central pathology review and DNA extraction, a total of 31 pure mucinous cases from Institut J. Bordet Biobank were included (referred to as IJB cohort). Tumor infiltrating lymphocytes (TILs) were assessed on hematoxylin and eosin (H&E). Low pass whole genome sequencing was conducted to assess ploidy and to detect copy number aberrations (CNAs). A second cohort was analyzed from a publicly available dataset (referred to as BRCA560 cohort), with available centrally reviewed histology subtyping and DNA methylation profiles (Nik-Zainal et al. Nature 2016). 207 ER+ primary BC, 14 of which were mucinous, had HumanMethylation450K methylation profiles. RNAseq was available for a subset of 145 cancers, 13 of which were mucinous. DNA methylation data was processed in R using the minfi package. Beta-values were normalized (preprocessQuantile) and dmpFinder was used to identify differentially methylated positions. Results In the IJB cohort, whole genome sequencing revealed that all but two tumors were diploid. The most frequently deleted cancer genes were RB1 (38,1%), CDH1 (23,8%), BRCA2 (38,1%),TP53 (23,8%), MAP2K4 (23,8%), EGFR (28,6%) and PGR (23,8%). In terms of amplifications, only ZNF217 (19,4%) and FGFR1/ZNF703 (9,5%) were observed. These mucinous tumors generally displayed low TIL levels (median=5%). In the BRCA560 cohort, there was no significant difference in the clinico-pathological features of ER+ IDC vs mucinous subtypes. At the genomic level, we identified a lower frequency of PIK3CA mutations as compared to ER+ IDC from the same cohort. At the DNA methylation level, we identified 8013 differentially methylated CpGs (q-val < .05) between IDC and mucinous tumors. The top differentially methylated CpG mapped to MUC2, an extracellular mucin. MUC2 was significantly demethylated in mucinous tumors as compared to IDC (q-val Conclusion To the best of our knowledge, this study is the first to report hypomethylation of MUC2 in mucinous carcinoma of the breast, as a possible mechanism of extracellular production of mucin in these tumors. Furthermore, it highlights the minor immune infiltration of these cancers, possibly prevented by the mucin, and sheds light on the few genomic alterations present in these overall stable cancer genomes. Citation Format: Desmedt C, Veys I, Nguyen B, Leduc S, Bareche Y, Majjaj S, Boeckx B, Lambrechts D, Sotiriou C. Molecular characterization of mucinous breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-07-04.

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

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

FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1-5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state., info:eu-repo/semantics/published

The development of immune checkpoint blockade (ICB) has changed the way we treat various cancers. While ICB produces durable survival benefits in a number of malignancies, a large proportion of treated patients do not derive clinical benefit. Recent clinical profiling studies have shed light on molecular features and mechanisms that modulate response to ICB. Nevertheless, none of these identified molecular features were investigated in large enough cohorts to be of clinical value.Literature review was carried out to identify relevant studies including clinical dataset of patients treated with ICB [anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) or the combination] and available sequencing data. Tumor mutational burden (TMB) and 37 previously reported gene expression (GE) signatures were computed with respect to the original publication. Biomarker association with ICB response (IR) and survival (progression-free survival/overall survival) was investigated separately within each study and combined together for meta-analysis.We carried out a comparative meta-analysis of genomic and transcriptomic biomarkers of IRs in over 3600 patients across 12 tumor types and implemented an open-source web application (predictIO.ca) for exploration. TMB and 21/37 gene signatures were predictive of IRs across tumor types. We next developed a de novo GE signature (PredictIO) from our pan-cancer analysis and demonstrated its superior predictive value over other biomarkers. To identify novel targets, we computed the T-cell dysfunction score for each gene within PredictIO and their ability to predict dual PD-1/CTLA-4 blockade in mice. Two genes, F2RL1 (encoding protease-activated receptor-2) and RBFOX2 (encoding RNA-binding motif protein 9), were concurrently associated with worse ICB clinical outcomes, T-cell dysfunction in ICB-naive patients and resistance to dual PD-1/CTLA-4 blockade in preclinical models.Our study highlights the potential of large-scale meta-analyses in identifying novel biomarkers and potential therapeutic targets for cancer immunotherapy.

Background: Immune checkpoint blockade (ICB) of PD-1 and/or CTLA-4 can induce long-lasting clinical responses in several neoplasias. However, despite the successful response in some cancer, it is still ineffective for other malignant lesions such as ovarian cancer. Studies on T cell dynamics demonstrated that antitumor immune response from ICB is mediated by clonal replacement of CD8 T cells. Moreover, the presence of clones expanded in both tumor and peripheral tissue, also called dual-expanded, can predict response to ICB. To understand T cell dynamics in ovarian cancer, we characterized intratumoral and peripheral immune cells by single-cell transcriptome and TCR-sequencing. Methods and results: Using the 5’ Chromium assay (10X Genomics) on CD45+ cells isolated from 5 naïve-treatment patients undergoing cytoreductive surgery, we identified T cell states and classified clones by location (blood or tumor) and expansion pattern. In addition, the data revealed that clones expanded within the tumor were terminally exhausted (upregulation of PDCD1, HAVCR2, CTLA4, ENTPD1, and CXCL13 genes), while dual-expanded clones (expanded in blood and tumor) upregulated genes associated with the cytotoxic function of CD8 T cells, such as granzymes (GZMA and GZMB) and perforin (PRF1). Considering the importance of dual-expanded clones in antitumor immune response, we interrogated the cellular interactions they encountered in the tumor microenvironment. Using the tool NicheNet we identified IL21 as one of the top ligands mediating the interaction between dual-expanded clones and T follicular helper-like CD4+ cells (Tfh-like cells). Interestingly, the gene signature of Tfh-like cells showed that besides dual-expanded clones, these cells might attract other immune cells through CXCL13 and oxysterol metabolites. We identified B cells and plasmacytoid dendritic cells (pDCs) as the major cell types recruited by the CXCL13-CXCR5 axis and oxysterol-GPR183, respectively. Additionally, memory-like CD8 T cells were found to express GPR183, suggesting that a gradient of oxysterol might recruit them to a region defined by Tfh-like cells. Conclusions: Taken together, our study provides insights into additional functions of Tfh-like cells in the recruitment of immune cells through oxysterol gradient. A better understanding of the T cell dynamics and antitumor immune response can provide potential development of novel immunotherapy for the treatment of ovarian cancer. Citation Format: Mayra Carneiro, Cheng Zhao, Paméla Thébault, Yacine Bareche, Kurosh Rahimi, Jean-François Cailhier, Anne-Marie Mes-Masson, John Stagg, Sophie Petropoulos, Réjean Lapointe. Crosstalk between peripherally expanded T cells and Tfh-like cells in shaping effector functions of CD8 T cells in ovarian 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 1361.

Recent efforts of gene expression profiling analyses recognized at least 4 different triple-negative breast cancer (TNBC) molecular subtypes. However, little is known regarding their tumor microenvironment (TME) heterogeneity., info:eu-repo/semantics/published

Alternative splicing (AS) plays a central role during cell-fate determination. However, how the core spliceosomal factors (CSFs) are involved in this process is poorly understood. Here, we report the down-regulation of the U2AF1 CSF during stem cell differentiation. To investigate its function in stemness and differentiation, we downregulated U2AF1 in human induced pluripotent stem cells (hiPSCs), using an inducible-shRNA system, to the level found in differentiated ectodermal, mesodermal and endodermal cells. RNA sequencing and computational analysis reveal that U2AF1 down-regulation modulates the expression of development-regulating genes and regulates transcriptional networks involved in cell-fate determination. Furthermore, U2AF1 down-regulation induces a switch in the AS of transcription factors (TFs) required to establish specific cell lineages, and favours the splicing of a differentiated cell-specific isoform of DNMT3B. Our results showed that the differential expression of the core spliceosomal factor U2AF1, between stem cells and the precursors of the three germ layers regulates a cell-type-specific alternative splicing programme and a transcriptional network involved in cell-fate determination via the modulation of gene expression and alternative splicing of transcription regulators.

Introduction: Triple-negative breast cancer (TNBC), representing about 10-20% of all breast cancers (BC), is a heterogeneous disease characterized by a clinically aggressive course, higher relapse rates and worse overall survival as compared to other BC subtypes. Recent efforts of genome-wide gene expression profiling have improved our understanding of the biological diversity of TNBCs reporting at least 6 different molecular subtypes namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). Little is known regarding the potential driving molecular events characterizing each subtype, their difference in survival and response to therapy. Moreover, limited progress has been made so far in the development of effective therapeutic strategies in TNBC partly due to the lack of obvious genetic targets. Further insight into the underlying genomic alterations is therefore needed. Aims: Here, we aimed to study the genomic aberrations that drive each of the TNBC molecular subtypes as defined by Lehmann et al. by applying an integrative analysis combining somatic mutation, copy number aberrations and gene expression profiles of 272 TNBC derived from METABRIC consortium. Methods: In silico analyses were performed using microarray, copy number aberrations profiles (CNA) and mutation profiles data retrieved from 272 TNBC patients from the METABRIC consortium. Somatic mutation profiles were derived from targeted sequencing of 173 cancer genes. CNAs at the gene level were computed using GISTIC 2.0.22. The relationship between TNBC molecular subtypes and genomic aberrations were analysed using Fisher test. Survival analyses were performed using Cox proportional hazard models adjust for standard clinical and pathologic variables. Results: Using a multivariate model, IM and M subtypes were significantly associated with good (HR=0.53; FDR=0.01) and poor (HR=1.8; FDR=0.01) prognosis respectively. BL1 subtype was found to be the most genomically instable subtype with high frequency of TP53 mutations (93%) and copy-number deletions in genes involved in DNA repair mechanisms (BRCA2, MDM2, RB1 and TP53) and MYC overexpression. BL2 subtype was enriched with TP53 (95%) and PIK3CA mutations (52%), whereas LAR subtype was characterized by high frequency of PI3KCA mutations (44%). Of interest, for these two subtypes, mutations in genes in the PI3K signalling pathway were mutually exclusive. IM subtype was enriched in NCOR2 mutations (13%), a co-repressor of the Notch pathway, associated with lower gene expression (p=0.035). M and MSL subtypes were significantly associated with higher signature scores for angiogenesis. In addition, M subtype was associated with higher frequency of NOTCH1 mutations (M p=2.4E-2). Conclusions: This integrative analysis combining somatic mutation, copy number and gene expression profiles shed new light on TNBC molecular heterogeneity, highlighting TNBC subtype-specific genomic aberrations which could potentially open new avenues for the development of effective targeted therapies in each TNBC molecular subtype such as DNA repair, Notch and angiogenesis. Citation Format: Bareche Y, Venet D, Rothe F, Sotiriou C. Uncovering TNBC molecular heterogeneity by applying integrative “omics” analyses with potential clinical implications [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-02.

Introduction: Triple negative breast cancer (TNBC) constitute 10-20% of all breast cancers and is associated with a worse prognosis and limited treatment options. Recent trials evaluating immune checkpoint blockade in TNBC demonstrated encouraging results for a subset of patients. TNBC is highly heterogeneous and its tumour microenvironment (TME) has been recognized as a critical determinant of its behavior and clinical outcome. Genome-wide gene expression profiling analyses have already improved our understanding of the complexity of this disease and have defined 6 different molecular subtypes namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR), exhibiting distinct biological and clinical characteristic. In this study, we aim to dissect the molecular diversity of the TME and more specifically to assess the immune landscape according to TNBC molecular subtypes. Methods: A cohort of 485 TNBC patient with publicly available data (RNA-Seq and Illumina HT-12 v3) from the METABRIC and the TCGA consortia were used in the gene expression analysis. Gene signatures reflecting different features or cellular components (immune, stromal, angiogenesis, lymphangiogenesis, hypoxia, metabolism) of the TME were used to evaluate multiple biological processes known to contribute to tumorogenesis. A compendium of 17 immune specific gene signatures and T cell localisation classification were used to evaluate the immune composition and spatial pattern of immune infiltrates. All parameters were compared using a logistic regression model to evaluate their relative contribution according to each molecular subtype. Results: Our analyses demonstrated that each molecular subtype exhibits different TME profiles, as well as specific immune composition and localisation. IM tumors were associated with the highest expression of immune-related gene signatures, enriched with adaptive immune cells and with a fully inflamed spatial pattern. MSL tumors were mostly associated with the expression of Lymphangiogenesis and Stromal TME signatures. They also exhibited some immune activity through the expression of immune gene signatures capturing innate immune and adaptive immunosuppressive cells. This subtype was mainly associated with margin restricted and to some extent with fully inflamed spatial pattern. BL1 tumors were associated with the expression of Metabolism TME signatures, along with fully inflamed and stroma restricted spatial pattern. To a lesser extent, this subtype was also associated with activated DC and CD4 Tem cells. LAR and M tumors exhibited an immune cold phenotype. They were associated with Stromal and Metabolism TME signatures, enriched in margin restricted spatial pattern and negatively associated with every immune cells. Conclusions: Our results demonstrate for the first time the huge heterogeneity that characterizes the TME of TNBCs. Identification of specific TME profiles could help to design more rationale and appropriate synergistic therapeutic combinations targeting TME elements in this high-risk disease. Citation Format: Bareche Y, Buisseret L, Gruosso T, Girard E, Venet D, Dupont F, Desmedt C, Park M, Rothé F, Stagg J, Sotiriou C. Unravelling triple-negative breast cancer tumor microenvironment heterogeneity using an integrative multiomic analysis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-03.

Expression of the ectonucleotidase CD73 by tumor cells, stromal cells, and immune cells is associated in cancer with immune suppression. In this study, we investigated the role of CD73 on the activity of the anti-HER2/ErbB2 monoclonal antibody (mAb) trastuzumab. In a prospective, randomized phase III clinical trial evaluating the activity of trastuzumab, high levels of CD73 gene expression were associated significantly with poor clinical outcome. In contrast, high levels of PD-1 and PD-L1 were associated with improved clinical outcome. In immunocompetent mouse models of HER2/ErbB2–driven breast cancer, CD73 expression by tumor cells and host cells significantly suppressed immune-mediated responses mediated by anti-ErbB2 mAb. Furthermore, anti-CD73 mAb therapy enhanced the activity of anti-ErbB2 mAb to treat engrafted or spontaneous tumors as well as lung metastases. Gene ontology enrichment analysis from gene-expression data revealed a positive association of CD73 expression with extracellular matrix organization, TGFβ genes, epithelial-to-mesenchymal transition (EMT) transcription factors and hypoxia-inducible-factor (HIF)-1 gene signature. Human mammary cells treated with TGFβ or undergoing EMT upregulated CD73 cell-surface expression, confirming roles for these pathways. In conclusion, our findings establish CD73 in mediating resistance to trastuzumab and provide new insights into how CD73 is regulated in breast cancer. Cancer Res; 77(20); 5652–63. ©2017 AACR.

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N

Background There is an urgent requirement to identify biomarkers to tailor treatment in human epidermal growth factor receptor 2 (HER2)-amplified early breast cancer treated with trastuzumab/pertuzumab-based chemotherapy. Methods Among the 225 patients randomly assigned to trastuzumab/pertuzumab concurrently or sequentially with an anthracycline-containing regimen or concurrently with an anthracycline-free regimen in the Tryphaena trial, we determined the percentage of tumor-infiltrating lymphocytes (TILs) at baseline in 213 patients, of which 126 demonstrated a pathological complete response (pCR; ypT0/is ypN0), with 28 demonstrating event-free survival (EFS) events. We investigated associations between baseline TIL percentage and either pCR or EFS after adjusting for clinicopathological characteristics using logistic and Cox regression models, respectively. To understand TIL biology, we evaluated associations between baseline TILs and baseline tumor gene expression data (800 gene set by NanoString) in a subset of 173 patients. All statistical tests were two-sided. Results Among the patients with measurable TILs at baseline, the median level was 14.1% (interquartile range = 7.1%-32.4%). After adjusting for clinicopathological characteristics, baseline percentage TIL was not associated with pCR (adjusted odds ratio [aOR] for every 10-percentage unit increase in TILs = 1.12, 95% confidence interval [CI] = 0.95 to 1.31, P = .17). At a median follow-up of 4.7 years, for every increase in baseline TILs of 10%, there was a 25% reduction in the hazard for an EFS event (aOR = 0.75, 95% CI = 0.56 to 1.00, P = .05) after adjusting for baseline clinicopathological characteristics and pCR. Additionally, genes associated with epithelial-mesenchymal transition, angiogenesis, and T-cell inhibition such as SNAIL1, ZEB1, NOTCH3, and B7-H3 were statistically significantly inversely correlated with percentage TIL. Conclusions Baseline TIL percentage provides independent prognostic information in patients treated with trastuzumab/pertuzumab-based neoadjuvant chemotherapy. However, further validation is required.

Background: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. Methods: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. Results: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01-0.56). Similar observations were made for cell type-specific quantifications (r = 0.001-0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64-0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52-0.82). Conclusions: There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL., SCOPUS: ar.j, info:eu-repo/semantics/published

BackgroundHydrolysis of extracellular ATP to adenosine (eADO) is an important immune checkpoint in cancer immunology. We here investigated the impact of the eADO pathway in high-grade serous ovarian cancer (HGSC) using multiparametric platforms.MethodsWe performed a transcriptomic meta-analysis of eADO-producing CD39 and CD73, an eADO signaling gene signature, immune gene signatures and clinical outcomes in approximately 1200 patients with HGSC. Protein expression, localization and prognostic impact of CD39, CD73 and CD8 were then performed on approximately 1000 cases on tissue microarray, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry and single-cell RNA sequencing on a subset of patients.ResultsConcomitant CD39 and CD73 gene expression, as well as high levels of an eADO gene signature, were associated with worse prognosis in patients with HGSC, notably in the immunoregulatory molecular subtype, characterized by an immune-active microenvironment. CD39 was further associated with primary chemorefractory and chemoresistant human HGSC and platinum-based chemotherapy of murine HGSC was significantly more effective in CD39-deficient mice. At protein level, CD39 and CD73 were predominantly expressed by cancer-associated fibroblasts, and CD39 was expressed on severely exhausted, clonally expanded and putative tissue-resident memory TILs.ConclusionsOur study revealed the clinical, immunological, subtype-specific impacts of eADO signaling in HGSC, unveiled the chemoprotective effect of CD39 and supports the evaluation of eADO-targeting agents in patients with ovarian cancer.

Although invasive ductal breast cancer (IDC) represents the most common histological type of breast cancer, minor subtypes exist such as mucinous breast cancer (MuBC). MuBC are distinguished by tumor cells floating in extracellular mucin. MuBC patients are generally older and associated with a favorable prognosis. To unravel the molecular architecture of MuBC, we applied low-pass whole-genome sequencing and microscopic evaluation of stromal tumor infiltrating lymphocytes to 30 MuBC from a retrospective institutional cohort. We further analyzed two independent datasets from the International Cancer Genomics Consortium and The Cancer Genome Atlas. Genomic data (n = 26 MuBC, n = 535 estrogen receptor [ER] positive/HER2-negative IDC), methylation data (n = 28 MuBC, n = 529 ER-positive/HER2-negative IDC), and transcriptomic data (n = 27 MuBC, n = 467 ER-positive/HER2-negative IDC) were analyzed. MuBC was characterized by low tumor infiltrating lymphocyte levels (median = 0.0%, average = 3.4%, 95% confidence interval = 1.9% to 4.9%). Compared with IDC, MuBC had a lower genomic instability (P =. 01, two-sided Mann-Whitney U test) and a decreased prevalence of PIK3CA mutations (39.7% in IDC vs 6.7% in MuBC, P =. 01 in the International Cancer Genomics Consortium; and 34.8% vs 0.0%, P =. 02 in The Cancer Genome Atlas, two-sided Fisher's exact test). Finally, our report identifies aberrant DNA methylation of MUC2 as a possible cause of extracellular production of mucin in MuBC., SCOPUS: ar.j, info:eu-repo/semantics/published

Recent efforts of genome-wide gene expression profiling analyses have improved our understanding of the biological complexity and diversity of triple negative breast cancers (TNBCs) reporting, at least 6 different molecular subtypes of TNBC namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). However, little is known regarding the potential driving molecular events within each subtype, their difference in survival and response to therapy. Further insight into the underlying genomic alterations is therefore needed., SCOPUS: ar.j, info:eu-repo/semantics/published

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

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

Background: Tumours are not only composed of malignant cells but also consist of a stromal micro-environment, which has been shown to influence cancer cell behaviour. Because the ageing process induces accumulation of senescent cells in the body, this micro-environment is thought to be different in cancers occurring in old patients compared with younger patients. More specifically, senescence-related fibroblastic features, such as the senescence-associated secretory profile (SASP) and the induction of autophagy, are suspected to stimulate tumour growth and progression. Methods: We compared gene expression profiles in stromal fields of breast carcinomas by performing laser capture microdissection of the cancer-associated stroma from eight old (aged ≥80 years at diagnosis) and nine young (aged, SCOPUS: ar.j, info:eu-repo/semantics/published

Purpose: Androgen receptor (AR) expression has been observed in about 70% of patients with breast cancer, but its prognostic role remains uncertain. Experimental Design: To assess the prognostic role of AR expression in early-stage breast cancer, we performed a meta-analysis of studies that evaluated the impact of AR at the protein and gene expression level on disease-free survival (DFS) and/or overall survival (OS). Eligible studies were identified by systematic review of electronic databases using the MeSH-terms “breast neoplasm” and “androgen receptor” and were selected after a qualitative assessment based on the REMARK criteria. A pooled gene expression analysis of 35 publicly available microarray data sets was also performed from patients with early-stage breast cancer with available gene expression and clinical outcome data. Results: Twenty-two of 33 eligible studies for the clinical meta-analysis, including 10,004 patients, were considered as evaluable for the current study after the qualitative assessment. AR positivity defined by IHC was associated with improved DFS in all patients with breast cancer [multivariate (M) analysis, HR 0.46; 95% confidence interval (CI) 0.37–0.58, P < 0.001] and better OS [M-HR 0.53; 95% CI, 0.38–0.73, P < 0.001]. Thirty-five datasets including 7,220 patients were eligible for the pooled gene expression analysis. High AR mRNA levels were found to confer positive prognosis overall in terms of DFS (HR 0.82; 95% CI 0.72–0.92;P = 0.0007) and OS (HR 0.84; 95% CI, 0.75–0.94; P = 0.02) only in univariate analysis. Conclusions: Our analysis, conducted among more than 17,000 women with early-stage breast cancer included in clinical and gene expression analysis, demonstrates that AR positivity is associated with favorable clinical outcome. Clin Cancer Res; 23(11); 2702–12. ©2016 AACR.

Several studies have demonstrated the feasibility of molecular screening of tumour samples for matching patients with cancer to targeted therapies. However, most of them have been carried out at institutional or national level. Herein, we report on the pilot phase of AURORA (NCT02102165), a European multinational collaborative molecular screening initiative for advanced breast cancer patients. Forty-one patients were prospectively enroled at four participating centres across Europe. Metastatic tumours were biopsied and profiled using an Ion Torrent sequencing platform at a central facility. Sequencing results were obtained for 63% of the patients in real-time with variable turnaround time stemming from delays between patient consent and biopsy. At least one clinically actionable mutation was identified in 73% of patients. We used the Illumina sequencing technology for orthogonal validation and achieved an average of 66% concordance of substitution calls per patient. Additionally, copy number aberrations inferred from the Ion Torrent sequencing were compared to single nucleotide polymorphism arrays and found to be 59% concordant on average. Although this study demonstrates that powerful next generation genomic techniques are logistically ready for international molecular screening programs in routine clinical settings, technical challenges remain to be addressed in order to ensure the accuracy and clinical utility of the genomic data., info:eu-repo/semantics/published

Bakground/objectives: Intense drug discovery efforts in the metabolic field highlight the need for novel strategies for the treatment of obesity. Alternative splicing (AS) and/or polyadenylation enable the LMNA gene to express distinct protein isoforms that exert opposing effects on energy metabolism and lifespan. Here we aimed to use the splicing factor SRSF 1 that contribute to the production of these different isoforms as a target to uncover new anti-obesity drug. Subjects/Methods: Small molecules modulating SR protein activity and splicing, we re tested for their abilities to interact with SRSF1 and to modulate LMNA (AS). Using an LMNA luciferase reporter we selected molecules that were tested in Diet Induced Obese (DIO) mice. Transcriptomic analyses were performed in the white adipose tissues from untreated and treated DIO mice and mice fed a chow diet. Results: We identified a small molecule that specifically interacted with the RS domain of SRSF1. ABX300 abolished diet induced obesity (DIO) in mice, leading to restoration of adipose tissue homeostasis. In contrast, ABX300 had no effect on mice fed a standard chow diet. A global transcriptomic analysis revealed similar profiles of White adipose tissue (WAT) from DIO mice treated with ABX300 and from untreated mice fed a chow diet. Mice treated with ABX300 exhibited an increase in O2 consumption and a switch in fuel preference toward lipids. Conclusions: Targeting SRSF1 with ABX300 compensates for changes in RNA biogenesis induced by fat accumulation and consequently represents a novel unexplored approach for the treatment of obesity.