NF-κB subunits RelA and c-Rel selectively control CD4+ T cell function in multiple sclerosis and cancer.

The outcome of cancer and autoimmunity is often dictated by the effector functions of CD4+ conventional T cells (Tconv). Although activation of the NF-κB signaling pathway has long been implicated in Tconv biology, the cell-autonomous roles of the separate NF-κB transcription-factor subunits are unknown. Here, we dissected the contributions of the canonical NF-κB subunits RelA and c-Rel to Tconv function. RelA, rather than c-Rel, regulated Tconv activation and cytokine production at steady-state and was required for polarization toward the TH17 lineage in vitro. Accordingly, RelA-deficient mice were fully protected against neuroinflammation in a model of multiple sclerosis due to defective transition to a pathogenic TH17 gene-expression program. Conversely, Tconv-restricted ablation of c-Rel impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. Moreover, Tconv required c-Rel for the response to PD-1-blockade therapy. Our data reveal distinct roles for canonical NF-κB subunits in different disease contexts, paving the way for subunit-targeted immunotherapies.

Spatial Transcriptomics Reveal Pitfalls and Opportunities for the Detection of Rare High-Plasticity Breast Cancer Subtypes.

Breast cancer is one of the most prominent types of cancers, in which therapeutic resistance is a major clinical concern. Specific subtypes, such as claudin-low and metaplastic breast carcinoma (MpBC), have been associated with high nongenetic plasticity, which can facilitate resistance. The similarities and differences between these orthogonal subtypes, identified by molecular and histopathological analyses, respectively, remain insufficiently characterized. Furthermore, adequate methods to identify high-plasticity tumors to better anticipate resistance are lacking. Here, we analyzed 11 triple-negative breast tumors, including 3 claudin-low and 4 MpBC, via high-resolution spatial transcriptomics. We combined pathological annotations and deconvolution approaches to precisely identify tumor spots, on which we performed signature enrichment, differential expression, and copy number analyses. We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia public databases for external validation of expression markers. By focusing our spatial transcriptomic analyses on tumor cells in MpBC samples, we bypassed the negative impact of stromal contamination and identified specific markers that are neither expressed in other breast cancer subtypes nor expressed in stromal cells. Three markers (BMPER, POPDC3, and SH3RF3) were validated in external expression databases encompassing bulk tumor material and stroma-free cell lines. We unveiled that existing bulk expression signatures of high-plasticity breast cancers are relevant in mesenchymal transdifferentiated compartments but can be hindered by abundant stromal cells in tumor samples, negatively impacting their clinical applicability. Spatial transcriptomic analyses constitute powerful tools to identify specific expression markers and could thus enhance diagnosis and clinical care of rare high-plasticity breast cancers.

Multiomic analysis of malignant pleural mesothelioma identifies molecular axes and specialized tumor profiles driving intertumor heterogeneity.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with rising incidence and challenging clinical management. Through a large series of whole-genome sequencing data, integrated with transcriptomic and epigenomic data using multiomics factor analysis, we demonstrate that the current World Health Organization classification only accounts for up to 10% of interpatient molecular differences. Instead, the MESOMICS project paves the way for a morphomolecular classification of MPM based on four dimensions: ploidy, tumor cell morphology, adaptive immune response and CpG island methylator profile. We show that these four dimensions are complementary, capture major interpatient molecular differences and are delimited by extreme phenotypes that-in the case of the interdependent tumor cell morphology and adapted immune response-reflect tumor specialization. These findings unearth the interplay between MPM functional biology and its genomic history, and provide insights into the variations observed in the clinical behavior of patients with MPM.

Gene signature of circulating platelet-bound neutrophils is associated with poor prognosis in cancer patients.

Beyond their critical role in hemostasis, platelets physically interact with neutrophils to form neutrophil-platelet aggregates (NPAs), enhancing neutrophil effector functions during inflammation. NPAs may also promote disease worsening in various inflammatory diseases. However, characterization of NPAs in cancer remains totally unexplored. Using ImageStreamX (ISX) imaging flow cytometer, we were not only allowed able to detect CD15+ CD14- CD36+ ITGA2B+ NPAs in both healthy donors' (HDs) and cancer patients' bloods, but we also showed that NPAs result from the binding of platelets preferentially to low-density neutrophils (LDNs) as opposed to normal-density neutrophils (NDNs). By reanalyzing two independent public scRNAseq data of whole blood leukocytes from cancer patients and HDs, we could identify a subset of neutrophils with high platelet gene expression that may correspond to NPAs. Moreover, we showed that cancer patients' derived NPAs possessed a distinct molecular signature compared to the other neutrophil subsets, independently of platelet genes. Gene ontology (GO) term enrichment analysis of this NPAs-associated neutrophil transcriptomic signature revealed a significant enrichment of neutrophil degranulation, chemotaxis and trans-endothelial migration GO terms. Lastly, using The Cancer Genome Atlas (TCGA), we could show by multivariate Cox analysis that the NPAs-associated neutrophil transcriptomic signature was associated with a worse patient prognosis in several cancer types. These results suggest that neutrophils from NPAs are systemically primed by platelets empowering them with cancer progression capacities once at tumor site. NPAs may therefore hold clinical utility as novel noninvasive blood prognostic biomarker in cancer patients with solid tumors.

Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure.

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence.

CD163+ tumor-associated macrophage accumulation in breast cancer patients reflects both local differentiation signals and systemic skewing of monocytes.

OBJECTIVES: The accumulation of tumor-associated macrophages (TAMs) is correlated with poor clinical outcome, but the mechanisms governing their differentiation from circulating monocytes remain unclear in humans. METHODS: Using multicolor flow cytometry, we evaluated TAMs phenotype in 93 breast cancer (BC) patients. Furthermore, monocytes from healthy donors were cultured in the presence of supernatants from dilacerated primary tumors to investigate their differentiation into macrophages (MΦ) in vitro. Additionally, we used transcriptomic analysis to evaluate BC patients' blood monocytes profiles. RESULTS: We observed that high intra-tumor CD163-expressing TAM density is predictive of reduced survival in BC patients. In vitro, M-CSF, TGF-ß and VEGF from primary tumor supernatants skewed the differentiation of healthy donor blood monocytes towards CD163highCD86lowIL-10high M2-like MΦ that strongly suppressed CD4+ T-cell expansion via PD-L1 and IL-10. In addition, blood monocytes from about 40% of BC patients displayed an altered response to in vitro stimulation, being refractory to type-1 MΦ (M1-MΦ) differentiation and secreting higher amounts of immunosuppressive, metastatic-related and angiogenic cytokines. Aside from showing that monocyte transcriptome is significantly altered by the presence of BC, we also demonstrated an overall metabolic de-activation in refractory monocytes of BC patients. In contrast, monocytes from sensitive BC patients undergoing normal M1-MΦ differentiation showed up-regulation of IFN-response genes and had no signs of metabolic alteration. CONCLUSION: Altogether, our results suggest that systemic factors skew BC patient blood monocytes towards a pro-metastatic profile, resulting in the accumulation of further polarised CD163high TAMs resembling type-2 MΦ (M2-MΦ) in the local BC microenvironment. These data indicate that monitoring circulating monocytes in BC patients may provide an indication of early systemic alterations induced by cancer and, thus, be instrumental in the development of improved personalised immunotherapeutic interventions.

Experimental and pan-cancer genome analyses reveal widespread contribution of acrylamide exposure to carcinogenesis in humans.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Relevance of a molecular tumour board (MTB) for patients' enrolment in clinical trials: experience of the Institut Curie.

BACKGROUND: High throughput molecular screening techniques allow the identification of multiple molecular alterations, some of which are actionable and can be targeted by molecularly targeted agents (MTA). We aimed at evaluating the relevance of using this approach in the frame of Institut Curie Molecular Tumor Board (MTB) to guide patients with cancer to clinical trials with MTAs. PATIENTS AND METHODS: We included all patients presented at Institut Curie MTB from 4 October 2014 to 31 October 2017. The following information was extracted from the chart: decision to perform tumour profiling, types of molecular analyses, samples used, molecular alterations identified and those which are actionable, and inclusion in a clinical trial with matched MTA. RESULTS: 736 patients were presented at the MTB. Molecular analyses were performed in 442 patients (60%). Techniques used included next-generation sequencing, comparative genomic hybridisation array and/or other techniques including immunohistochemistry in 78%, 51% and 58% of patients, respectively. Analyses were performed on a fresh frozen biopsy in 91 patients (21%), on archival tissue (fixed or frozen) in 326 patients (74%) and on both archival and fresh frozen biopsy in 25 patients (6%). At least one molecular alteration was identified in 280 analysed patients (63%). An actionable molecular alteration was identified in 207 analysed patients (47%). Forty-five analysed patients (10%) were enrolled in a clinical trial with matched MTA and 29 additional patients were oriented and included in a clinical trial based on a molecular alteration identified prior to the MTB analysis. Median time between date of specimen reception and molecular results was 28 days (range: 5-168). CONCLUSIONS: The implementation of an MTB at Institut Curie enabled the inclusion of 10% of patients into a clinical trial with matched therapy.

Integrative genomic profiling of large-cell neuroendocrine carcinomas reveals distinct subtypes of high-grade neuroendocrine lung tumors.

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: "type I LCNECs" with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and "type II LCNECs" enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors.

Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Genome-scale mutational signatures of aflatoxin in cells, mice, and human tumors.

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene-this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.

TP53 Variations in Human Cancers: New Lessons from the IARC TP53 Database and Genomics Data.

TP53 gene mutations are one of the most frequent somatic events in cancer. The IARC TP53 Database (http://p53.iarc.fr) is a popular resource that compiles occurrence and phenotype data on TP53 germline and somatic variations linked to human cancer. The deluge of data coming from cancer genomic studies generates new data on TP53 variations and attracts a growing number of database users for the interpretation of TP53 variants. Here, we present the current contents and functionalities of the IARC TP53 Database and perform a systematic analysis of TP53 somatic mutation data extracted from this database and from genomic data repositories. This analysis showed that IARC has more TP53 somatic mutation data than genomic repositories (29,000 vs. 4,000). However, the more complete screening achieved by genomic studies highlighted some overlooked facts about TP53 mutations, such as the presence of a significant number of mutations occurring outside the DNA-binding domain in specific cancer types. We also provide an update on TP53 inherited variants including the ones that should be considered as neutral frequent variations. We thus provide an update of current knowledge on TP53 variations in human cancer as well as inform users on the efficient use of the IARC TP53 Database.

MutSpec: a Galaxy toolbox for streamlined analyses of somatic mutation spectra in human and mouse cancer genomes.

BACKGROUND: The nature of somatic mutations observed in human tumors at single gene or genome-wide levels can reveal information on past carcinogenic exposures and mutational processes contributing to tumor development. While large amounts of sequencing data are being generated, the associated analysis and interpretation of mutation patterns that may reveal clues about the natural history of cancer present complex and challenging tasks that require advanced bioinformatics skills. To make such analyses accessible to a wider community of researchers with no programming expertise, we have developed within the web-based user-friendly platform Galaxy a first-of-its-kind package called MutSpec. RESULTS: MutSpec includes a set of tools that perform variant annotation and use advanced statistics for the identification of mutation signatures present in cancer genomes and for comparing the obtained signatures with those published in the COSMIC database and other sources. MutSpec offers an accessible framework for building reproducible analysis pipelines, integrating existing methods and scripts developed in-house with publicly available R packages. MutSpec may be used to analyse data from whole-exome, whole-genome or targeted sequencing experiments performed on human or mouse genomes. Results are provided in various formats including rich graphical outputs. An example is presented to illustrate the package functionalities, the straightforward workflow analysis and the richness of the statistics and publication-grade graphics produced by the tool. CONCLUSIONS: MutSpec offers an easy-to-use graphical interface embedded in the popular Galaxy platform that can be used by researchers with limited programming or bioinformatics expertise to analyse mutation signatures present in cancer genomes. MutSpec can thus effectively assist in the discovery of complex mutational processes resulting from exogenous and endogenous carcinogenic insults.

Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.

BACKGROUND: Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urologic cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA-sequencing studies using fresh-frozen tumors. METHODS: Here, we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multisample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of AA nephropathy. RESULTS: LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of approximately 10×. Analysis at 3 to 9× coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern, whereas 83 cancer driver genes were enriched for recurrent nonsynonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding. CONCLUSIONS: LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas. IMPACT: By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures.

Revealing the Molecular Portrait of Triple Negative Breast Tumors in an Understudied Population through Omics Analysis of Formalin-Fixed and Paraffin-Embedded Tissues.

Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer.

Renal cell carcinomas of chronic kidney disease patients harbor the mutational signature of carcinogenic aristolochic acid.

Aristolochic acid (AA) is a potent dietary cytotoxin and carcinogen, and an established etiological agent underlying severe human nephropathies and associated upper urinary tract urothelial cancers, collectively designated aristolochic acid nephropathy (AAN). Its genome-wide mutational signature, marked by predominant A:T > T:A transversions occurring in the 5'-CpApG-3' trinucleotide context and enriched on the nontranscribed gene strand, has been identified in human upper urinary tract urothelial carcinomas from East Asian patients and in experimental systems. Here we report a whole-exome sequencing screen performed on DNA from formalin-fixed, paraffin-embedded renal cell carcinomas (RCC) arising in chronic renal disease patients from a Balkan endemic nephropathy (EN) region. In the EN regions, the disease results from the consumption of bread made from wheat contaminated by seeds of Aristolochia clematitis, an AA-containing plant. In five of eight (62.5%) tested RCC tumor specimens, we observed the characteristic global mutational signature consistent with the mutagenic effects of AA. This signature was absent in the control RCC samples obtained from patients from a nonendemic, metropolitan region. By identifying a new tumor type associated with the AA-driven genome-wide mutagenic process in the context of renal disease, our results suggest new epidemiological and public health implications for the RCC incidence worldwide, particularly for the high-risk regions with unregulated use of AA-containing traditional herbal medicines.

Modelling mutational landscapes of human cancers in vitro.

Experimental models that recapitulate mutational landscapes of human cancers are needed to decipher the rapidly expanding data on human somatic mutations. We demonstrate that mutation patterns in immortalised cell lines derived from primary murine embryonic fibroblasts (MEFs) exposed in vitro to carcinogens recapitulate key features of mutational signatures observed in human cancers. In experiments with several cancer-causing agents we obtained high genome-wide concordance between human tumour mutation data and in vitro data with respect to predominant substitution types, strand bias and sequence context. Moreover, we found signature mutations in well-studied human cancer driver genes. To explore endogenous mutagenesis, we used MEFs ectopically expressing activation-induced cytidine deaminase (AID) and observed an excess of AID signature mutations in immortalised cell lines compared to their non-transgenic counterparts. MEF immortalisation is thus a simple and powerful strategy for modelling cancer mutation landscapes that facilitates the interpretation of human tumour genome-wide sequencing data.