Deciphering the spatial landscape and plasticity of immunosuppressive fibroblasts in breast cancer.

Although heterogeneity of FAP+ Cancer-Associated Fibroblasts (CAF) has been described in breast cancer, their plasticity and spatial distribution remain poorly understood. Here, we analyze trajectory inference, deconvolute spatial transcriptomics at single-cell level and perform functional assays to generate a high-resolution integrated map of breast cancer (BC), with a focus on inflammatory and myofibroblastic (iCAF/myCAF) FAP+ CAF clusters. We identify 10 spatially-organized FAP+ CAF-related cellular niches, called EcoCellTypes, which are differentially localized within tumors. Consistent with their spatial organization, cancer cells drive the transition of detoxification-associated iCAF (Detox-iCAF) towards immunosuppressive extracellular matrix (ECM)-producing myCAF (ECM-myCAF) via a DPP4- and YAP-dependent mechanism. In turn, ECM-myCAF polarize TREM2+ macrophages, regulatory NK and T cells to induce immunosuppressive EcoCellTypes, while Detox-iCAF are associated with FOLR2+ macrophages in an immuno-protective EcoCellType. FAP+ CAF subpopulations accumulate differently according to the invasive BC status and predict invasive recurrence of ductal carcinoma in situ (DCIS), which could help in identifying low-risk DCIS patients eligible for therapeutic de-escalation.

Residual ANTXR1+ myofibroblasts after chemotherapy inhibit anti-tumor immunity via YAP1 signaling pathway.

Although cancer-associated fibroblast (CAF) heterogeneity is well-established, the impact of chemotherapy on CAF populations remains poorly understood. Here we address this question in high-grade serous ovarian cancer (HGSOC), in which we previously identified 4 CAF populations. While the global content in stroma increases in HGSOC after chemotherapy, the proportion of FAP+ CAF (also called CAF-S1) decreases. Still, maintenance of high residual CAF-S1 content after chemotherapy is associated with reduced CD8+ T lymphocyte density and poor patient prognosis, emphasizing the importance of CAF-S1 reduction upon treatment. Single cell analysis, spatial transcriptomics and immunohistochemistry reveal that the content in the ECM-producing ANTXR1+ CAF-S1 cluster (ECM-myCAF) is the most affected by chemotherapy. Moreover, functional assays demonstrate that ECM-myCAF isolated from HGSOC reduce CD8+ T-cell cytotoxicity through a Yes Associated Protein 1 (YAP1)-dependent mechanism. Thus, efficient inhibition after treatment of YAP1-signaling pathway in the ECM-myCAF cluster could enhance CD8+ T-cell cytotoxicity. Altogether, these data pave the way for therapy targeting YAP1 in ECM-myCAF in HGSOC.

Molecular underpinnings and environmental drivers of loss of heterozygosity in Drosophila intestinal stem cells.

During development and aging, genome mutation leading to loss of heterozygosity (LOH) can uncover recessive phenotypes within tissue compartments. This phenomenon occurs in normal human tissues and is prevalent in pathological genetic conditions and cancers. While studies in yeast have defined DNA repair mechanisms that can promote LOH, the predominant pathways and environmental triggers in somatic tissues of multicellular organisms are not well understood. Here, we investigate mechanisms underlying LOH in intestinal stem cells in Drosophila. Infection with the pathogenic bacteria, Erwinia carotovora carotovora 15, but not Pseudomonas entomophila, increases LOH frequency. Using whole genome sequencing of somatic LOH events, we demonstrate that they arise primarily via mitotic recombination. Molecular features and genetic evidence argue against a break-induced replication mechanism and instead support cross-over via double Holliday junction-based repair. This study provides a mechanistic understanding of mitotic recombination, an important mediator of LOH, and its effects on stem cells in vivo.

Imaging and multi-omics datasets converge to define different neural progenitor origins for ATRT-SHH subgroups.

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.

Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma.

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

The mutational landscape of skull base and spinal chordomas and the identification of potential prognostic and theranostic biomarkers.

OBJECTIVE: Chordomas are rare bone neoplasms characterized by a high recurrence rate and no benefit from any approved medical treatment to date. However, the investigation of molecular alterations in chordomas could be essential to prognosticate, guide clinical decision-making, and identify theranostic biomarkers. The aim of this study was to provide a detailed genomic landscape of a homogeneous series of 64 chordoma samples, revealing driver events, theranostic markers, and outcome-related genomic features. METHODS: The authors conducted whole-exome sequencing (WES), targeted next-generation sequencing, and RNA sequencing of 64 skull base and spinal chordoma samples collected between December 2006 and September 2020. Clinical, histological, and radiological data were retrospectively analyzed and correlated to genetic findings. RESULTS: The authors identified homozygous deletions of CDKN2A/2B, PIK3CA mutations, and alterations affecting genes of SWI/SNF chromatin remodeling complexes (PBRM1 and ARID1A) as potential theranostic biomarkers. Using matched germline WES, they observed a higher frequency of a common genetic variant (rs2305089; p.(Gly177Asp)) in TBXT (97.8%, p < 0.001) compared to its distribution in the general population. PIK3CA mutation was identified as an independent biomarker of short progression-free survival (HR 10.68, p = 0.0008). Loss of CDKN2A/2B was more frequently observed in spinal tumors and recurrent tumors. CONCLUSIONS: In the current study, the authors identified driver events such as PBRM1 and PIK3CA mutations, TBXT alterations, or homozygous deletions of CDKN2A/2B, which could, for some, be considered potential theranostic markers and could allow for identifying novel therapeutic approaches. With the aim of a future biomolecular prognostication classification, alterations affecting PIK3CA and CDKN2A/2B could be considered as poor prognostic biomarkers.

Epigenetically controlled tumor antigens derived from splice junctions between exons and transposable elements.

Oncogenesis often implicates epigenetic alterations, including derepression of transposable elements (TEs) and defects in alternative splicing. Here, we explore the possibility that noncanonical splice junctions between exons and TEs represent a source of tumor-specific antigens. We show that mouse normal tissues and tumor cell lines express wide but distinct ranges of mRNA junctions between exons and TEs, some of which are tumor specific. Immunopeptidome analyses in tumor cell lines identified peptides derived from exon-TE splicing junctions associated to MHC-I molecules. Exon-TE junction-derived peptides were immunogenic in tumor-bearing mice. Both prophylactic and therapeutic vaccinations with junction-derived peptides delayed tumor growth in vivo. Inactivation of the TE-silencing histone 3-lysine 9 methyltransferase Setdb1 caused overexpression of new immunogenic junctions in tumor cells. Our results identify exon-TE splicing junctions as epigenetically controlled, immunogenic, and protective tumor antigens in mice, opening possibilities for tumor targeting and vaccination in patients with cancer.

Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma.

BACKGROUND: High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS: We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS: We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS: Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment.

Dichotomy in Neutralizing Antibody Induction to Peptide-Conjugated Vaccine in Squalene Emulsion Contrast With Aluminum Hydroxide Formulation.

W614A-3S peptide is a modified 3S motif of the HIV-gp41 (mutation W614A). We previously detected the presence of natural neutralizing antibodies directed against W614A-3S peptide (NAbs) in long-term non-progressor HIV+ patients. Here, we compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in inducing broadly-NAbs (bNAbs). Rabbit and mouse models were used to screen the induction of bNAbs following 4 immunizations. SQE was more efficient than Alum formulation in inducing W614A-3S-specific bNAbs with up to 67%-93% of HIV strains neutralized. We then analyzed the quality of peptide-specific murine B cells by single-cell gene expression by quantitative reverse transcription-PCR and single-cell V(D)J sequencing. We found more frequent germinal center B cells in SQE than in Alum, albeit with a different gene expression profile. The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations. All sixteen BCR sequences which were cloned were specific of peptide. Adjuvant formulations of W614A-3S-peptide-conjugated immunogen impact the quantity and quality of B cell immune responses at both the gene expression level and BCR sequence.

PD-L1 and ICOSL discriminate human Secretory and Helper dendritic cells in cancer, allergy and autoimmunity.

Dendritic cells (DC) are traditionally classified according to their ontogeny and their ability to induce T cell response to antigens, however, the phenotypic and functional state of these cells in cancer does not necessarily align to the conventional categories. Here we show, by using 16 different stimuli in vitro that activated DCs in human blood are phenotypically and functionally dichotomous, and pure cultures of type 2 conventional dendritic cells acquire these states (termed Secretory and Helper) upon appropriate stimuli. PD-L1highICOSLlow Secretory DCs produce large amounts of inflammatory cytokines and chemokines but induce very low levels of T helper (Th) cytokines following co-culturing with T cells. Conversely, PD-L1lowICOSLhigh Helper DCs produce low levels of secreted factors but induce high levels and a broad range of Th cytokines. Secretory DCs bear a single-cell transcriptomic signature indicative of mature migratory LAMP3+ DCs associated with cancer and inflammation. Secretory DCs are linked to good prognosis in head and neck squamous cell carcinoma, and to response to checkpoint blockade in Melanoma. Hence, the functional dichotomy of DCs we describe has both fundamental and translational implications in inflammation and immunotherapy.

H3K27me3 conditions chemotolerance in triple-negative breast cancer.

The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.

Tissue-resident FOLR2+ macrophages associate with CD8+ T cell infiltration in human breast cancer.

Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2+ tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2+ macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8+ T cells. FOLR2+ macrophages efficiently prime effector CD8+ T cells ex vivo. The density of FOLR2+ macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies.

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

Transcriptional Programs Define Intratumoral Heterogeneity of Ewing Sarcoma at Single-Cell Resolution.

EWSR1-FLI1, the chimeric oncogene specific for Ewing sarcoma (EwS), induces a cascade of signaling events leading to cell transformation. However, it remains elusive how genetically homogeneous EwS cells can drive the heterogeneity of transcriptional programs. Here, we combine independent component analysis of single-cell RNA sequencing data from diverse cell types and model systems with time-resolved mapping of EWSR1-FLI1 binding sites and of open chromatin regions to characterize dynamic cellular processes associated with EWSR1-FLI1 activity. We thus define an exquisitely specific and direct enhancer-driven EWSR1-FLI1 program. In EwS tumors, cell proliferation and strong oxidative phosphorylation metabolism are associated with a well-defined range of EWSR1-FLI1 activity. In contrast, a subpopulation of cells from below and above the intermediary EWSR1-FLI1 activity is characterized by increased hypoxia. Overall, our study reveals sources of intratumoral heterogeneity within EwS tumors.

FLASH Irradiation Spares Lung Progenitor Cells and Limits the Incidence of Radio-induced Senescence.

PURPOSE: One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. "FLASH" irradiation at very high dose-rate is a new treatment modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity. EXPERIMENTAL DESIGN: Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc-/- mice exposed to bilateral thorax irradiation as well as human lung cells grown in vitro. RESULTS: In vitro studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc-/- mice harboring critically short telomeres and lack of telomerase activity. CONCLUSIONS: The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.

Clonally Expanded T Cells Reveal Immunogenicity of Rhabdoid Tumors.

Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.

Decentralization of Next-Generation RNA Sequencing-Based MammaPrint® and BluePrint® Kit at University Hospitals Leuven and Curie Institute Paris.

A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting.

Circulating tumor DNA analysis enables molecular characterization of pediatric renal tumors at diagnosis.

Circulating tumor DNA (ctDNA) is a powerful tool for the molecular characterization of cancer. The most frequent pediatric kidney tumors (KT) are Wilms' tumors (WT), but other diagnoses may occur. According to the SIOP strategy, in most countries pediatric KT have a presumptive diagnosis of WT if they are clinically and radiologically compatible. The histologic confirmation is established after post-chemotherapy nephrectomy. Thus, there is a risk for a small fraction of patients to receive neoadjuvant chemotherapy that is not adapted to the disease. The aim of this work is to perform molecular diagnosis of pediatric KT by tumor genetic characterization based on the analysis of ctDNA. We analyzed ctDNA extracted from plasma samples of 18 pediatric patients with KT by whole-exome sequencing and compared the results to their matched tumor and germline DNA. Copy number alterations (CNAs) and single nucleotide variations (SNVs) were analyzed. We were able to detect tumor cell specific genetic alterations-CNAs, SNVs or both-in ctDNA in all patients except in one (for whom the plasma sample was obtained long after nephrectomy). These results open the door to new applications for the study of ctDNA with regards to the molecular diagnosis of KT, with a possibility of its usefulness for adapting the treatment early after diagnosis, but also for disease monitoring and follow up.

Targeted next-generation sequencing identifies clinically relevant somatic mutations in a large cohort of inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of primary breast cancer. Using a custom-made breast cancer gene sequencing panel, we investigated somatic mutations in IBC to better understand the genomic differences compared with non-IBC and to consider new targeted therapy in IBC patients. METHODS: Targeted next-generation sequencing (NGS) of 91 candidate breast cancer-associated genes was performed on 156 fresh-frozen breast tumor tissues from IBC patients. Mutational profiles from 197 primary breast tumors from The Cancer Genome Atlas (TCGA) were used as non-IBC controls for comparison analysis. The mutational landscape of IBC was correlated with clinicopathological data and outcomes. RESULTS: After genotype calling and algorithmic annotations, we identified 392 deleterious variants in IBC and 320 variants in non-IBC cohorts, respectively. IBC tumors harbored more mutations than non-IBC (2.5 per sample vs. 1.6 per sample, p < 0.0001). Eighteen mutated genes were significantly different between the two cohorts, namely TP53, CDH1, NOTCH2, MYH9, BRCA2, ERBB4, POLE, FGFR3, ROS1, NOTCH4, LAMA2, EGFR, BRCA1, TP53BP1, ESR1, THBS1, CASP8, and NOTCH1. In IBC, the most frequently mutated genes were TP53 (43.0%), PIK3CA (29.5%), MYH9 (8.3%), NOTCH2 (8.3%), BRCA2 (7.7%), ERBB4 (7.1%), FGFR3 (6.4%), POLE (6.4%), LAMA2 (5.8%), ARID1A (5.1%), NOTCH4 (5.1%), and ROS1 (5.1%). After grouping 91 genes on 10 signaling pathways, we found that the DNA repair pathway for the triple-negative breast cancer (TNBC) subgroup, the RTK/RAS/MAPK and cell cycle pathways for the HR-/HER2+ subgroup, the DNA repair, RTK/RAS/MAPK, and NOTCH pathways for the HR+/HER2- subgroup, and the DNA repair, epigenome, and diverse pathways for the HR+/HER2+ subgroup were all significantly differently altered between IBC and non-IBC. PIK3CA mutation was independently associated with worse metastasis-free survival (MFS) in IBC since the median MFS for the PIK3CA mutant type was 26.0 months and for the PIK3CA wild type was 101.1 months (p = 0.002). This association was observed in TNBC (p = 0.04) and the HR-/HER2+ subgroups (p = 0.0003), but not in the HR+/HER2- subgroup of IBC. CONCLUSIONS: Breast cancer-specific targeted NGS uncovered a high frequency of deleterious somatic mutations in IBC, some of which may be relevant for clinical management.

Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway.

Presentation of exogenous antigens on MHC-I molecules, termed cross-presentation, is essential for cytotoxic CD8+ T cell responses. In mice, dendritic cells (DCs) that arise from monocytes (mo-DCs) during inflammation have a key function in these responses by cross-presenting antigens locally in peripheral tissues. Whether human naturally-occurring mo-DCs can cross-present is unknown. Here, we use human mo-DCs and macrophages directly purified from ascites to address this question. Single-cell RNA-seq data show that ascites CD1c+ DCs contain exclusively monocyte-derived cells. Both ascites mo-DCs and monocyte-derived macrophages cross-present efficiently, but are inefficient for transferring exogenous proteins into their cytosol. Inhibition of cysteine proteases, but not of proteasome, abolishes cross-presentation in these cells. We conclude that human monocyte-derived cells cross-present exclusively using a vacuolar pathway. Finally, only ascites mo-DCs provide co-stimulatory signals to induce effector cytotoxic CD8+ T cells. Our findings thus provide important insights on how to harness cross-presentation for therapeutic purposes.