The NF-κB RelA transcription factor is not required for CD8+ T-cell function in acute viral infection and cancer.

CD8+ T cells are critical mediators of pathogen clearance and anti-tumor immunity. Although signaling pathways leading to the activation of NF-κB transcription factors have crucial functions in the regulation of immune responses, the CD8+ T cell-autonomous roles of the different NF-κB subunits, are still unresolved. Here, we investigated the function of the ubiquitously expressed transcription factor RelA in CD8+ T-cell biology using a novel mouse model and gene-edited human cells. We found that CD8+ T cell-specific ablation of RelA markedly altered the transcriptome of ex vivo stimulated cells, but maintained the proliferative capacity of both mouse and human cells. In contrast, in vivo experiments showed that RelA deficiency did not affect the CD8+ T-cell response to acute viral infection or transplanted tumors. Our data suggest that in CD8+ T cells, RelA is dispensable for their protective activity in pathological contexts.

Added value of whole-exome and RNA sequencing in advanced and refractory cancer patients with no molecular-based treatment recommendation based on a 90-gene panel.

INTRODUCTION: The objective was to determine the added value of comprehensive molecular profile by whole-exome and RNA sequencing (WES/RNA-Seq) in advanced and refractory cancer patients who had no molecular-based treatment recommendation (MBTR) based on a more limited targeted gene panel (TGP) plus array-based comparative genomic hybridization (aCGH). MATERIALS AND METHODS: In this retrospective analysis, we selected 50 patients previously included in the PROFILER trial (NCT01774409) for which no MBT could be recommended based on a targeted 90-gene panel and aCGH. For each patient, the frozen tumor sample mirroring the FFPE sample used for TGP/aCGH analysis were processed for WES and RNA-Seq. Data from TGP/aCGH were reanalyzed, and together with WES/RNA-Seq, findings were simultaneously discussed at a new molecular tumor board (MTB). RESULTS: After exclusion of variants of unknown significance, a total of 167 somatic molecular alterations were identified in 50 patients (median: 3 [1-10]). Out of these 167 relevant molecular alterations, 51 (31%) were common to both TGP/aCGH and WES/RNA-Seq, 19 (11%) were identified by the TGP/aCGH only and 97 (58%) were identified by WES/RNA-Seq only, including two fusion transcripts in two patients. A MBTR was provided in 4/50 (8%) patients using the information from TGP/aCGH versus 9/50 (18%) patients using WES/RNA-Seq findings. Three patients had similar recommendations based on TGP/aCGH and WES/RNA-Seq. CONCLUSIONS: In advanced and refractory cancer patients in whom no MBTR was recommended from TGP/aCGH, WES/RNA-Seq allowed to identify more alterations which may in turn, in a limited fraction of patients, lead to new MBTR.

Isocitrate dehydrogenase wt and IDHmut adult-type diffuse gliomas display distinct alterations in ribosome biogenesis and 2'O-methylation of ribosomal RNA.

BACKGROUND: High-grade adult-type diffuse gliomas (HGGs) constitute a heterogeneous group of aggressive tumors that are mostly incurable. Recent advances highlighting the contribution of ribosomes to cancer development have offered new clinical perspectives. Here, we uncovered that isocitrate dehydrogenase (IDH)wt and IDHmut HGGs display distinct alterations of ribosome biology, in terms of rRNA epitranscriptomics and ribosome biogenesis, which could constitute novel hallmarks that can be exploited for the management of these pathologies. METHODS: We analyzed (1) the ribosomal RNA 2'O-ribose methylation (rRNA 2'Ome) using RiboMethSeq and in-house developed bioinformatics tools (https://github.com/RibosomeCRCL/ribomethseq-nfandrRMSAnalyzer) on 3 independent cohorts compiling 71 HGGs (IDHwt n = 30, IDHmut n = 41) and 9 non-neoplastic samples, (2) the expression of ribosome biogenesis factors using medium throughput RT-qPCR as a readout of ribosome biogenesis, and (3) the sensitivity of 5 HGG cell lines to RNA Pol I inhibitors (CX5461, BMH-21). RESULTS: Unsupervised analysis demonstrated that HGGs could be distinguished based on their rRNA 2'Ome epitranscriptomic profile, with IDHwt glioblastomas displaying the most significant alterations of rRNA 2'Ome at specific sites. In contrast, IDHmut HGGs are largely characterized by an overexpression of ribosome biogenesis factors compared to non-neoplastic tissues or IDHwt glioblastomas. Finally, IDHmut HGG-derived spheroids display higher cytotoxicity to CX5461 than IDHwt glioblastoma, while all HGG spheroids display a similar cytotoxicity to BMH-21. CONCLUSIONS: In HGGs, IDH mutational status is associated with specific alterations of the ribosome biology and with distinct sensitivities to RNA Pol I inhibitors.

Dissecting the Origin of Heterogeneity in Uterine and Ovarian Carcinosarcomas.

Gynecologic carcinosarcomas (CS) are biphasic neoplasms composed of carcinomatous (C) and sarcomatous (S) malignant components. Because of their rarity and histologic complexity, genetic and functional studies on CS are scarce and the mechanisms of initiation and development remain largely unknown. Whole-genome analysis of the C and S components reveals shared genomic alterations, thus emphasizing the clonal evolution of CS. Reconstructions of the evolutionary history of each tumor further reveal that C and S samples are composed of both ancestral cell populations and component-specific subclones, supporting a common origin followed by distinct evolutionary trajectories. However, while we do not find any recurrent genomic features associated with phenotypic divergence, transcriptomic and methylome analyses identify a common mechanism across the cohort, the epithelial-to-mesenchymal transition (EMT), suggesting a role for nongenetic factors in inflicting changes to cellular fate. Altogether, these data accredit the hypothesis that CS tumors are driven by both clonal evolution and transcriptomic reprogramming, essential for susceptibility to transdifferentiation upon encountering environmental cues, thus linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences. Significance: We have provided a detailed characterization of the genomic landscape of CS and identified EMT as a common mechanism associated with phenotypic divergence, linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences.

Pembrolizumab in Lymphopenic Metastatic Breast Cancer Patients Treated with Metronomic Cyclophosphamide: A Clinical and Translational Prospective Study.

Purpose: Metastatic endocrine-resistant breast cancer (MBC) is a disease with poor prognosis and few treatment options. Low lymphocyte count is associated with limited overall survival. In a prospective cohort of lymphopenic patients with HER-2 negative MBC, we assessed the clinical and biological impact of pembrolizumab combined with metronomic cyclophosphamide. Experimental Design: This multicenter Phase II study evaluated the safety and clinical activity of pembrolizumab (intravenous (IV), 200mg, every 3 weeks) combined with metronomic cyclophosphamide (50mg/day, per os) in lymphopenic adult patients with HER2-negative MBC previously treated by at least one line of chemotherapy in this setting according to a Simon's minimax two-stage design. Blood and tumor samples were collected to assess the impact of the combined treatment on circulating immune cells and the tumor immune microenvironment through multiparametric flow cytometry and multiplex immunofluorescence analyses. Primary endpoint was the clinical benefit rate at 6 months of treatment (CBR-6M). Secondary endpoints were objective response rate (ORR), duration of response, progression free survival (PFS), and overall survival (OS). Results: Two out of the twenty treated patients presented clinical benefit (one Tumor Mutational Burden (TMB)-high patient with complete response (CR) and one patient with objective response (OR) per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST V1.1) associated with a strong increase of cytokine-producing and proliferating CD4+ T cells and higher CD8+ T cells to macrophage ratios in the tumor. This impact on CD4+ and CD8+ T cell polyfunctionality was still observed more than one year for the patient with CR. A decreased in their absolute number of CD4+ and CD8+ memory T cells was observed in other patients. Conclusion: Pembrolizumab combined with metronomic cyclophosphamide was well tolerated, and displayed limited anti-tumoral activity in lymphopenic MBC. Correlative translational data of our trial advocates for additional studies with other chemotherapy combinations.

Genetic Analysis of Lung Cancer and the Germline Impact on Somatic Mutation Burden.

BACKGROUND: Germline genetic variation contributes to lung cancer (LC) susceptibility. Previous genome-wide association studies (GWAS) have implicated susceptibility loci involved in smoking behaviors and DNA repair genes, but further work is required to identify susceptibility variants. METHODS: To identify LC susceptibility loci, a family history-based genome-wide association by proxy (GWAx) of LC (48 843 European proxy LC patients, 195 387 controls) was combined with a previous LC GWAS (29 266 patients, 56 450 controls) by meta-analysis. Colocalization was used to explore candidate genes and overlap with existing traits at discovered susceptibility loci. Polygenic risk scores (PRS) were tested within an independent validation cohort (1 666 LC patients vs 6 664 controls) using variants selected from the LC susceptibility loci and a novel selection approach using published GWAS summary statistics. Finally, the effects of the LC PRS on somatic mutational burden were explored in patients whose tumor resections have been profiled by exome (n = 685) and genome sequencing (n = 61). Statistical tests were 2-sided. RESULTS: The GWAx-GWAS meta-analysis identified 8 novel LC loci. Colocalization implicated DNA repair genes (CHEK1), metabolic genes (CYP1A1), and smoking propensity genes (CHRNA4 and CHRNB2). PRS analysis demonstrated that these variants, as well as subgenome-wide significant variants related to expression quantitative trait loci and/or smoking propensity, assisted in LC genetic risk prediction (odds ratio = 1.37, 95% confidence interval = 1.29 to 1.45; P < .001). Patients with higher genetic PRS loads of smoking-related variants tended to have higher mutation burdens in their lung tumors. CONCLUSIONS: This study has expanded the number of LC susceptibility loci and provided insights into the molecular mechanisms by which these susceptibility variants contribute to LC development.

PD-1 Blockade in Solid Tumors with Defects in Polymerase Epsilon.

Missense mutations in the polymerase epsilon (POLE) gene have been reported to generate proofreading defects resulting in an ultramutated genome and to sensitize tumors to checkpoint blockade immunotherapy. However, many POLE-mutated tumors do not respond to such treatment. To better understand the link between POLE mutation variants and response to immunotherapy, we prospectively assessed the efficacy of nivolumab in a multicenter clinical trial in patients bearing advanced mismatch repair-proficient POLE-mutated solid tumors. We found that only tumors harboring selective POLE pathogenic mutations in the DNA binding or catalytic site of the exonuclease domain presented high mutational burden with a specific single-base substitution signature, high T-cell infiltrates, and a high response rate to anti-PD-1 monotherapy. This study illustrates how specific DNA repair defects sensitize to immunotherapy. POLE proofreading deficiency represents a novel agnostic biomarker for response to PD-1 checkpoint blockade therapy. SIGNIFICANCE: POLE proofreading deficiency leads to high tumor mutational burden with high tumor-infiltrating lymphocytes and predicts anti-PD-1 efficacy in mismatch repair-proficient tumors. Conversely, tumors harboring POLE mutations not affecting proofreading derived no benefit from PD-1 blockade. POLE proofreading deficiency is a new tissue-agnostic biomarker for cancer immunotherapy. This article is highlighted in the In This Issue feature, p. 1397.

Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

Comprehensive characterization of claudin-low breast tumors reflects the impact of the cell-of-origin on cancer evolution.

Claudin-low breast cancers are aggressive tumors defined by the low expression of key components of cellular junctions, associated with mesenchymal and stemness features. Although they are generally considered as the most primitive breast malignancies, their histogenesis remains elusive. Here we show that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups that emerge from unique evolutionary processes. Genetic, gene methylation and gene expression analyses reveal that two of the subgroups relate, respectively, to luminal breast cancers and basal-like breast cancers through the activation of an EMT process over the course of tumor progression. The third subgroup is closely related to normal human mammary stem cells. This unique subgroup of breast cancers shows a paucity of genomic aberrations and a low frequency of TP53 mutations, supporting the emerging notion that the intrinsic properties of the cell-of-origin constitute a major determinant of the genetic history of tumorigenesis.

Partially methylated domains are hypervariable in breast cancer and fuel widespread CpG island hypermethylation.

Global loss of DNA methylation and CpG island (CGI) hypermethylation are key epigenomic aberrations in cancer. Global loss manifests itself in partially methylated domains (PMDs) which extend up to megabases. However, the distribution of PMDs within and between tumor types, and their effects on key functional genomic elements including CGIs are poorly defined. We comprehensively show that loss of methylation in PMDs occurs in a large fraction of the genome and represents the prime source of DNA methylation variation. PMDs are hypervariable in methylation level, size and distribution, and display elevated mutation rates. They impose intermediate DNA methylation levels incognizant of functional genomic elements including CGIs, underpinning a CGI methylator phenotype (CIMP). Repression effects on tumor suppressor genes are negligible as they are generally excluded from PMDs. The genomic distribution of PMDs reports tissue-of-origin and may represent tissue-specific silent regions which tolerate instability at the epigenetic, transcriptomic and genetic level.

Mutational Profile of Aggressive, Localised Prostate Cancer from African Caribbean Men Versus European Ancestry Men.

Causes of high mortality of prostate cancer in men of African ancestry living in the French West Indies are still debated, between suspicions of environmental factors and genetic susceptibility. We report an integrated genomic study of 25 tumour tissues from radical prostatectomy of aggressive (defined by International Society of Urological Pathology ≥3) prostate cancer patients (10 African Caribbean and 15 French Caucasian) using single nucleotide polymorphism arrays, whole-genome sequencing, and RNA sequencing. The results show that African Caribbean tumours are characterised by a more frequent deletion at 1q41-43 encompassing the DNA repair gene PARP1, and a higher proportion of intrachromosomal rearrangements including duplications associated with CDK12 truncating mutations. Transcriptome analyses show an overexpression of genes related to androgen receptor activity in African Caribbean tumours, and of PVT1, a long non-coding RNA located at 8q24 that confirms the strong involvement of this region in prostate tumours from men of African ancestry. Patient summary: Mortality of prostate cancer is higher in African Caribbean men than in French Caucasian men. Specificities of the former could be explained by genomic events linked with key genes such as DNA damage pathway genes PARP1, CDK12, and the oncogenic long non-coding RNA gene PVT1 at the 8q24 prostate cancer susceptibility locus.

[Contribution and challenges of Big Data in oncology]. / Apport et défis des Big Data en cancérologie.

Since the first draft of the human genome sequence published in 2001, the cost of sequencing has dramatically decreased. The development of new technologies such as next generation sequencing led to a comprehensive characterization of a large number of tumors of various types as well as to significant advances in precision medicine. Despite the valuable information this technological revolution has allowed to produce, the vast amount of data generated resulted in the emergence of new challenges for the biomedical community, such as data storage, processing and mining. Here, we describe the contribution and challenges of Big Data in oncology.

A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers.

HER2-positive breast cancer has long proven to be a clinically distinct class of breast cancers for which several targeted therapies are now available. However, resistance to the treatment associated with specific gene expressions or mutations has been observed, revealing the underlying diversity of these cancers. Therefore, understanding the full extent of the HER2-positive disease heterogeneity still remains challenging. Here we carry out an in-depth genomic characterization of 64 HER2-positive breast tumour genomes that exhibit four subgroups, based on the expression data, with distinctive genomic features in terms of somatic mutations, copy-number changes or structural variations. The results suggest that, despite being clinically defined by a specific gene amplification, HER2-positive tumours melt into the whole luminal-basal breast cancer spectrum rather than standing apart. The results also lead to a refined ERBB2 amplicon of 106 kb and show that several cases of amplifications are compatible with a breakage-fusion-bridge mechanism.

A phenotypic and mechanistic perspective on heterogeneity of HER2-positive breast cancers.

Analysis of gene expression and whole-genome features of 64 human epidermal growth factor 2 (HER2)-positive breast tumors supports the idea that their intrinsic heterogeneity actually reflects their cell of origin, suggesting that HER2 amplification is an embedded event in the natural history of these tumors. Possible mechanisms for this event involve breakage-fusion-bridge and chromothripsis.

Microwave-Assisted Superheating and/or Microwave-Specific Superboiling (Nucleation-Limited Boiling) of Liquids Occurs under Certain Conditions but is Mitigated by Stirring.

Temporary superheating and sustained nucleation-limited "superboiling" of unstirred liquids above the normal atmospheric boiling point have been documented during microwave heating. These phenomena are reliably observed under prescribed conditions, although the duration (of superheating) and magnitude (of superheating and superboiling) vary according to system parameters such as volume of the liquid and the size and shape of the vessel. Both phenomena are mitigated by rapid stirring with an appropriate stir bar and/or with the addition of boiling chips, which provide nucleation sites to support the phase-change from liquid to gas. With proper experimental design and especially proper stirring, the measured temperature of typical organic reaction mixtures heated at reflux will be close to the normal boiling point temperature of the solvent, whether heated using microwave radiation or conventional convective heat transfer. These observations are important to take into consideration when comparing reaction rates under conventional and microwave heating.

Microwave-specific acceleration of a Friedel-Crafts reaction: evidence for selective heating in homogeneous solution.

Thermally promoted Friedel-Crafts benzylation of arene solvents has been examined under both conventional convective heating with an oil bath and heating using microwave (MW) energy. Bulk solution temperatures-as measured by internal and external temperature probes and as defined by solvent reflux-were comparable in both sets of experiments. MW-specific rate enhancements were documented under certain conditions and not others. The observed rate enhancements at a given temperature are proposed to arise from selective MW heating of polar solutes, perturbing thermal equilibrium between the solute and bulk solution. Central to MW-specific thermal phenomena is the difference between heat and temperature. Temperature is a measure of the ensemble average kinetic molecular energy of all solution components, but temperature does not provide information about solute-specific energy differences that may arise as a consequence of selective MW heating. Enhanced chemical reactivity of the MW-absorbing solute can be described as a MW-specific "extra-temperature thermal effect", because the measurable solution temperature only captures a portion of the solute kinetic molecular energy. Experimental factors that favor MW-specific rate enhancements are discussed with an eye toward future development of MW-actuated organic reactions, in which the observed thermal reactivity exceeds what is predicted from temperature-based Arrhenius calculations.

UMD-MLH1/MSH2/MSH6 databases: description and analysis of genetic variations in French Lynch syndrome families.

Lynch syndrome is an autosomal dominant disease caused by germ line heterozygous mutations mainly involving the MSH2, MLH1 and MSH6 genes that belong to the DNA MisMatch Repair (MMR) genes family. The French network counting the 16 licensed laboratories involved in Lynch syndrome genetic testing developed three locus-specific databases with the UMD software (www.umd.be/MLH1/, www.umd.be/MSH2/ and www.umd.be/MSH6/) that presently contain a total of 7047 sequence variations including 707 distinct variations of a priori unknown functional significance (VUS) that were identified through complete mutation screening or targeted predictive testing. Mutation carriers are at high risk for developing early-onset colorectal and endometrial adenocarcinomas. Consensus clinical guidelines have been proposed, allowing the efficient detection of curable lesions. The major challenge of genetic testing is to reliably classify the genomic variations in those patients who seek genetic counseling. Combining the interactive tools of the software, the relevant published data and mainly original information produced by the French MisMatch Repair network, the UMD-MLH1/MSH2/MSH6 databases provide interpretation data for the 707 VUS that were classified according to the IARC 5-Class system. These public databases are regularly updated to improve the classification of all registered VUS, exploring their role in cancer pre-disposition based on structural and functional approaches.

A seven-gene signature aggregates a subgroup of stage II colon cancers with stage III.

Colorectal cancer is one of the most common cancers in the world. Histological staging is efficient, but combination with molecular markers may improve tumor classification. Gene expression profiles have been defined as prognosis predictors among stage II and III tumors, but their implementation in medical practice remains controversial. Stage II tumors have been recognized as a heterogeneous group, and high-risk morphologic features have been used to justify adjuvant chemotherapy. We propose here the investigation of clinical features and expression profiles from stage II and stage III colon carcinomas without DNA mismatch repair defects. Two series of 130 and 66 colon cancer samples were obtained. Expression profiles were established on oligonucleotide microarrays and processed in the R/Bioconductor environment. Hierarchical, then supervised, analyses were successively performed by applying a data-sampling approach. A molecular signature of seven genes was found to cluster stage III tumors with adjusted p values lower than 10(-10). A subgroup of stage II tumors aggregated this cluster in both series. No correlation was found with disease severity, but the function of the discriminating genes suggests that tumors have been classified according to their putative response to adjuvant targeted or classic therapies. Further pharmacogenetic studies might verify this observation.

8q24 Cancer risk allele associated with major metastatic risk in inflammatory breast cancer.

BACKGROUND: Association studies have identified low penetrance alleles that participate to the risk of cancer development. The 8q24 chromosomal region contains several such loci involved in various cancers that have been recently studied for their propensity to influence the clinical outcome of prostate cancer. We investigated here two 8q24 breast and colon cancer risk alleles in the close vicinity of the MYC gene for their role in the occurrence of distant metastases. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective series of 449 patients affected with breast or colon adenocarcinoma was genotyped for the rs13281615 and/or rs6983267 SNPs. Statistical analyses were done using the survival package v2.30 in the R software v2.9.1. The two SNPs did not influence the development of distant metastases of colon cancer; rs6983267 showed a mild effect on breast cancer. However, this effect was greatly emphasized when considering inflammatory breast cancer (IBC) solely. Replicated on a larger and independent series of IBC the contribution of the genotype to the metastatic risk of IBC was found an independent predictor of outcome (p = 2e-4; OR 8.3, CI95:2.6-33). CONCLUSIONS/SIGNIFICANCE: Our study shows first that the monitoring of this specific germline variation may add a substantial tool for IBC prognostication, an aggressive disease that evolves towards distant metastases much more frequently than non-IBC and for which no reliable prognostic factor is available in medical practice. Second, it more generally suggests that risk alleles, while associated with low susceptibility, could correlate with a high risk of metastasis.