Search

Breast cancer is one of the most prominent types of cancers, in which therapeutic resistance is still a major clinical hurdle. Specific subtypes like Claudin-low (CL) and metaplastic breast cancers (MpBC) have been associated with high non-genetic plasticity, which can facilitate resistance. The overlaps and differences between these orthogonal subtypes, respectively identified by molecular and histopathological analyses, are however still insufficiently characterised. Adequate methods to identify high-plasticity tumours to better anticipate resistance are furthermore still lacking. Here we analysed 11 triple negative breast tumours, including 3 CL and 4 MpBC samples,viahigh-resolution spatial transcriptomics. We combined pathological annotations and deconvolution approaches to precisely identify tumour spots, on which we performed signature enrichment, differential expression and copy-number analyses. We used the TCGA and CCLE public databases for external validation of expression markers. By levying spatial transcriptomics to focus analyses only to tumour cells in MpBC samples, and therefore bypassing the negative impact of stromal contamination, we could identify specific markers that are not expressed in other subtypes nor stromal cells. Three markers (BMPER, POPDC3andSH3RF3) could furthermore be validated in external expression databases encompassing bulk tumour material and stroma-free cell lines. We find that existing bulk expression signatures of high-plasticity breast cancers are relevant in mesenchymal transdifferentiated compartments but can be hindered by stromal cell prevalence in tumour samples, negatively impacting their clinical applicability. Spatial transcriptomics analyses can however help identify more specific expression markers, and could thus enhance diagnosis and clinical care of rare high-plasticity breast cancers.

Le français haïtien (FH) est une variété de parler francophone propre à Haïti et différente à bien des égards des autres variétés de parlers issues de la même matrice qu’est le français historique. Arrivé au milieu de la première moitié du XVIIe siècle dans l’espace qui est devenu Haïti aujourd’hui, le français va évoluer en se différenciant des variétés repérables à l’époque en France continentale et de celles qui vont se développer dans d’autres colonies françaises implantées dans la Caraïbe et ailleurs au cours de la même période (ou presque). Le contact de langues étant une source de variations dans le fonctionnement de toute langue, le FH se développera au contact du créole haïtien à l’émergence duquel il va contribuer, au contact de langues africaines, mais aussi à celui de l’anglo-américain et de l’espagnol. Les spécificités du FH proviennent de normes endogènes qui se développent dans la pratique communautaire de la langue mais aussi du contact de langues sus-évoqué. La conscience des normes endogènes est présente chez les enseignants de français mais la question de la normativisation du FH n’est pas posée. L’une des raisons pouvant expliquer cela est que les Haïtiens vivent le français comme une langue extérieure à leur communauté et que, par conséquent, ils ne seraient pas autorisés à intervenir sur son corpus.

Background: Our goal was to identify a gene-expression-based surrogate of genomic instability (GI) associated with the transformation of oral potentially malignant disorder (OPMD) into oral squamous cell carcinoma (OSCC). Methods: GI was defined as the fraction of genome altered (FGA). Training sets included the CCLE and TCGA databases. The relevance of the enrichment score of the top correlated genes, referred to as the GIN score, was evaluated in eight independent public datasets from the GEO repository, including a cohort of patients with OPMD with available outcome. Results: A set of 20 genes correlated with FGA in head and neck SCC were identified. A significant correlation was found between the 20-gene based GIN score and FGA in 95 esophagus SCC (r = 0.59) and 501 lung SCC (r = 0.63), and in 33 OPMD/OSCC (r = 0.38). A significantly increased GIN score was observed at different stages of oral carcinogenesis (normal–dysplasia –OSCC) in five independent datasets. The GIN score was higher in 10 OPMD that transformed into oral cancer compared to 10 nontransforming OPMD (p = 0.0288), and was associated with oral-cancer-free survival in 86 patients with OPMD (p = 0.0081). Conclusions: The GIN score is a gene-expression surrogate of GI, and is associated with oral carcinogenesis and OPMD malignant transformation.

Oral potentially malignant disorders (OPMD) may precede oral squamous cell carcinoma (OSCC). Reported rates of malignant transformation of OPMD range from 3 to 50%. While some clinical, histological, and molecular factors have been associated with a high-risk OPMD, they are, to date, insufficiently accurate for treatment decision-making. Moreover, this range highlights differences in the clinical definition of OPMD, variation in follow-up periods, and molecular and biological heterogeneity of OPMD. Finally, while treatment of OPMD may improve outcome, standard therapy has been shown to be ineffective to prevent OSCC development in patients with OPMD. In this perspective paper, several experts discuss the main challenges in oral cancer prevention, in particular the need to (i) to define an OPMD classification system by integrating new pathological and molecular characteristics, aiming (ii) to better identify OPMD at high risk of malignant transformation, and (iii) to develop treatment strategies to eradicate OPMD or prevent malignant transformation.

The evolutionary dynamics of tumor initiation remain undetermined, and the interplay between neoplastic cells and the immune system is hypothesized to be critical in transformation. Colorectal cancer (CRC) presents a unique opportunity to study the transition to malignancy as pre-cancers (adenomas) and early stage cancers are frequently detected and surgically removed. Here, we demonstrate a key role for the immune response in tumor initiation by studying tumor-immune eco-evolutionary dynamics from pre-cancer to carcinoma using a computational model, ecological analysis of digital pathology data, and multi-region exome sequencing and neoantigen prediction in a total of 62 patient samples. Modelling indicates there are several potential routes to malignancy, each of which uniquely sculpts tumor ecology and intra-tumor antigenic heterogeneity (aITH). In patient samples, the immune microenvironment was characterized using the spatial distribution of 17 markers across registered whole-slide images, and patterns of intra-lesion aITH measured using multi-region exome sequencing and neoantigen prediction. The patient data were best described by a model whereby immunogenic adenomas do not progress to CRC because they are under immune control, and progression initially proceeds in adenomas with low immunogenicity followed by the gradual construction of an immunosuppressive niche depleted in CD8+ cytotoxic T cells. There was little evidence for immune blockade (PD-L1 expression) in tumor initiation or progression. These results suggest that re-engineering the immunosuppressive niche may prove to be an effective immunotherapy in CRC.

Aneuploidy, defined as the loss and gain of whole and part chromosomes, is a near-ubiquitous feature of cancer genomes, is prognostic, and likely an important determinant of cancer cell biology. In colorectal cancer (CRC), aneuploidy is found in virtually all tumours, including precursor adenomas. However, the temporal evolutionary dynamics that select for aneuploidy remain broadly uncharacterised. Here we perform genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that cross-sectionally represent the distinct stages of tumour evolution, and longitudinally track individual tumours through metastasis and treatment. Precancer lesions (adenomas) exhibited low levels of aneuploidy but high intra-tumour heterogeneity, whereas cancers had high aneuploidy but low heterogeneity, indicating that progression is through a genetic bottleneck that suppresses diversity. Individual CRC glands from the same tumour have similar karyotypes, despite prior evidence of ongoing instability at the cell level. Pseudo-stable aneuploid genomes were observed in metastatic lesions sampled from liver and other organs, after chemo- or targeted therapies, and late recurrences detected many years after the diagnosis of a primary tumour. Modelling indicates that these data are consistent with the action of stabilising selection that ‘traps’ cancer cell genomes on a fitness peak defined by the specific pattern of aneuploidy. These data show that the initial progression of CRC requires the traversal of a rugged fitness landscape and subsequent genomic evolution, including metastatic dissemination and therapeutic resistance, is constrained by stabilising selection.

Summary Despite advances in single-cell and molecular techniques, it is still unclear how to best quantify phenotypic heterogeneity in cancer cells that evolved beyond normal, known classifications. We present an approach to phenotypically characterize cells based on their activities rather than static classifications. We validated the detectability of specific activities (epithelial-mesenchymal transition, glycolysis) in single cells, using targeted RT-qPCR analyses and in vitro inductions. We analyzed 50 established activity signatures as a basis for phenotypic description in public data and computed cell-cell distances in 28,513 cells from 85 patients and 8 public datasets. Despite not relying on any classification, our measure correlated with standard diversity indices in populations of known structure. We identified bottlenecks as phenotypic diversity reduced upon colorectal cancer initiation. This suggests that focusing on what cancer cells do rather than what they are can quantify phenotypic diversity in universal fashion, to better understand and predict intra-tumor heterogeneity dynamics., Graphical Abstract, Highlights • Cells categorized as having the same identity can perform different activities • Single-cell expression data can be used to infer the activities cells take part in • Activity profiles provide a basis to measure phenotypic cell-cell divergence • Cell activity can quantify intra-tumor heterogeneity more fully than identity, Biological Sciences; Mathematical Biosciences; Cancer Systems Biology; Cancer

Bladder cancer is the 9th most commonly diagnosed cancer. Nearly half of patients with early stage bladder cancer treated with the immune-stimulating agent BCG have disease recurrence, while 13% progress to invasive bladder cancer. Here we explored the potential of tumor mutational heterogeneity and the role of pro- and anti-inflammatory cytokines to identify different subtypes of bladder cancer that may predict therapeutic response to BCG. Further, we used mathematical modeling of dosing strategies to infer tumor response to varying doses and time schedules f BCG administration. As a proof-of-concept, present adaptive therapy scheduling of BCG as a viable strategy to control tumor size and minimize recurrence.

Our previous work demonstrated a key function of the thyroid hormone nuclear receptor TRα1, a T3-modulated transcription factor, in controlling intestinal development and homeostasis via the Wnt and Notch pathways. Importantly, increased expression of TRα1 in the intestinal epithelium in a mutated Apc genetic background (vil-TRα1/Apc+/1638N mice) accelerated tumorigenesis and contributed to a more aggressive tumor phenotype compared to that of the Apc mutants alone. Therefore, the aim of this study was to determine the relevance of this synergistic effect in human colorectal cancers and to gain insights into the mechanisms involved. We analyzed cohorts of patients by in silico and experimental approaches and observed increased TRα1 expression and a significant correlation between TRα1 levels and Wnt activity. TRα1 loss-of-function and gain-of-function in Caco2 cell lines not only confirmed that TRα1 levels control Wnt activity but also demonstrated the role of TRα1 in regulating cell proliferation and migration. Finally, upon investigation of the molecular mechanisms responsible for the Wnt-TRα1 association, we described the repression by TRα1 of several Wnt inhibitors, including Frzb, Sox17 and Wif1. In conclusion, our results underline an important functional interplay between the thyroid hormone nuclear receptor TRα1 and the canonical Wnt pathway in intestinal cancer initiation and progression. More importantly, we show for the first time that the expression of TRα1 is induced in human colorectal cancers.

Ce texte met un trajet personnel, professionnel et scientifique, en regard avec celui qu’a connu depuis plusieurs decennies la Didactique des Langues Etrangeres et Secondes. Dans un meme mouvement, des points de vue – epistemologie, disciplines, ingenierie, contextes – et des horizons linguistiques et culturels de nature a les eclairer sont presentes. Trois revolutions ont impacte le domaine : sciences du langage, sciences humaines et sociales, technosciences cognitives et informatiques. Mon hypothese actuelle est que seule une didactique de mise en reseaux (« reticulaire »), appuyee sur le numerique, peut structurer une education en langues integrative et coherente.

The low risk of progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma can lead to over-diagnosis and over-treatment of BE patients. This may be addressed through a better understanding of the dynamics surrounding BE malignant progression. Although genetic diversity has been characterized as a marker of malignant development, it is still unclear how BE arises and develops. Here we uncover the evolutionary dynamics of BE at crypt and biopsy levels in eight individuals, including four patients that experienced malignant progression. We assay eight individual crypts and the remaining epithelium by SNP array for each of 6–11 biopsies over 2 time points per patient (358 samples in total). Our results indicate that most Barrett’s segments are clonal, with similar number and inferred rates of alterations observed for crypts and biopsies. Divergence correlates with geographical location, being higher near the gastro-esophageal junction. Relaxed clock analyses show that genomic instability precedes and is enhanced by genome doubling. These results shed light on the clinically relevant evolutionary dynamics of BE., Clonal dynamics of Barrett’s esophagus (BE) leading to cancer are poorly understood. Here, they report BE segments are clonal, have frequent mutations at the gastro-esophageal junction, genomic instability precedes genome doubling/clonal expansion, and a correlation between inter- and intra-biopsy genetic diversity.

Intra-tumour genetic heterogeneity (ITH) fosters drug resistance and is a critical hurdle to clinical treatment. ITH can be well-measured using multi-region sampling but this is costly and challenging to implement. There is therefore a need for tools to estimate ITH in individual samples, using standard genomic data such as SNP-arrays, that could be implemented routinely. We designed two novel scores S and R, respectively based on the Shannon diversity index and Ripley’s L statistic of spatial homogeneity, to quantify ITH in single SNP-array samples. We created in-silico and in-vitro mixtures of tumour clones, in which diversity was known for benchmarking purposes. We found significant but highly-variable associations of our scores with diversity in-silico (p in–vitro (p = 0.015 and p = 0.085). Our scores were also correlated to previous ITH estimates from sequencing data but heterogeneity in the fraction of tumour cells present across samples hampered accurate quantification. The prognostic potential of both scores was moderate but significantly predictive of survival in several tumour types (corrected p = 0.03). Our work thus shows how individual SNP-arrays reveal intra-sample clonal diversity with moderate accuracy.

Heparan sulfate (HS) 6-O-endosulfatases (Sulfs) have emerged recently as critical regulators of many physiological and pathological processes. By removing 6-O-sulfates from specific HS sequences, they modulate the activities of a variety of growth factors and morphogens, including fibroblast growth factor (FGF)-1. However, little is known about the functions of Sulfs in inflammation. Tumour-necrosis factor (TNF)-α plays an important role in regulating the behaviour of fibroblasts. In this study, we examined the effect of this inflammatory cytokine on the expression of Sulfs in human MRC-5 fibroblasts. Compositional analysis of HS from TNF-α-treated cells showed a strong reduction in the amount of the trisulfated UA2S-GlcNS6S disaccharide, which suggested a selective reaction of 6-O-desulfation. Real-time PCR analysis revealed that TNF-α increased Sulf-1 expression in a dose- and time-dependent manner, via a mechanism involving NF-ĸB, ERK1/2 and p38 MAPK. In addition, we confirmed that cell stimulation with TNF-α was accompanied by the secretion of an active form of Sulf-1. To study the function of Sulf- 1, we examined the responses induced by FGF-1. We showed that ERK1/2 activation and cell proliferation were markedly reduced in TNF-α-treated MRC-5 cells compared with untreated cells. Silencing the expression of Sulf-1 by RNA interference restored the responses induced by FGF-1, which indicated that TNF-α-mediated induction of the sulfatase indeed resulted in alterations of HS biological properties. Taken together, our results indicate that Sulf-1 is responsive to TNF-α stimulation and may function as an autocrine regulator of fibroblast expansion in the course of an inflammatory response.

Colorectal cancer develops from its precursor lesion, the adenoma. The immune system is hypothesized to be key in modulating progression, but tumor-immune eco-evolutionary dynamics remain uncharacterized. Here, we demonstrate a key role for immune evasion in the progression of human benign disease to colorectal cancer. We constructed a mathematical model of tumor-immune eco-evolutionary dynamics that predicted ecological succession, from an "immune-hot" adenoma immune ecology rich in T cells to an "immune-cold" carcinoma ecology, deficient in T cells and rich in immunosuppressive cells. Using a cross-sectional cohort of adenomas and carcinomas, we validated this prediction by direct measurement of the tumor-immune ecology using whole-slide 10-marker immunohistochemistry (IHC), and analysis of neoantigen clonal architecture multi-region exome sequencing data. Changes in immune ecology relax selection against antigens with high recognition potentials. This study indicates that immune surveillance represents a key evolutionary bottleneck in the evolution of colon cancer.

Heparan sulfate (HS) is recognized as an important player in a wide range of dynamic steps of inflammatory reactions. Thereby, structural HS remodeling is likely to play an important role in the regulation of inflammatory and immune responses; however, little is known about underlying mechanism. In this study, we analyzed the regulation of expression of HS 3-O-sulfotransferases (HS3STs) in response to inflammatory stimuli. We found that among the seven HS3ST isoenzymes, only the expression of HS3ST3B was markedly up-regulated in human primary monocytes and the related cell line THP1 after exposure to TLR agonists. TNF-α was also efficient, to a lesser extent, to increase HS3ST3B expression, while IL-6, IL-4, and IFN-γ were poor inducers. We then analyzed the molecular mechanisms that regulate the high expression of HS3ST3B in response to LPS. Based on the expression of HS3ST3B transcripts and on the response of a reporter gene containing the HS3ST3B1 promoter, we provide evidence that LPS induces a rapid and strong transcription of HS3ST3B1 gene, which was mainly dependent on the activation of NF-κB and JNK signaling pathways. Additionally, active p38 MAPK and de novo synthesized proteins are involved in post-transcriptional mechanisms to maintain a high level of HS3ST3B mRNA to a steady state. Altogether, our findings indicate that HS3ST3B1 gene behaves as a primary response gene, suggesting that it may play an important role in making 3-O-sulfated HS with specific functions in the regulation of inflammatory and immune responses. J. Cell. Biochem. 117: 1529-1542, 2016. © 2015 Wiley Periodicals, Inc.

Cancer is a potentially lethal disease, in which patients with nearly identical genetic backgrounds can develop a similar pathology through distinct combinations of genetic alterations. We aimed to reconstruct the evolutionary process underlying tumour initiation, using the combination of convergence and discrepancies observed across 2,742 cancer genomes from 9 tumour types. We developed a framework using the repeatability of cancer development to score the local malignant adaptation (LMA) of genetic clones, as their potential to malignantly progress and invade their environment of origin. Using this framework, we found that pre-malignant skin and colorectal lesions appeared specifically adapted to their local environment, yet insufficiently for full cancerous transformation. We found that metastatic clones were more adapted to the site of origin than to the invaded tissue, suggesting that genetics may be more important for local progression than for the invasion of distant organs. In addition, we used network analyses to investigate evolutionary properties at the system-level, highlighting that different dynamics of malignant progression can be modelled by such a framework in tumour-type-specific fashion. We find that occurrence-based methods can be used to specifically recapitulate the process of cancer initiation and progression, as well as to evaluate the adaptation of genetic clones to given environments. The repeatability observed in the evolution of most tumour types could therefore be harnessed to better predict the trajectories likely to be taken by tumours and pre-neoplastic lesions in the future.

The University laboratories are places where teaching, extension and research activities are develop, which harmful substances are manipulated and hazardous waste are generated, the lack of information about this makes them an inadequate provision causing human health and environmental risks. This research proposes the implementation of environmental education as an alternative for waste management and safety in the University of Magdalena laboratories. Applying a series of polls showed the effectiveness with efficiency or assertively rises at 30% cognitive level during the process. It recommends to obtain better results is necessary evaluate the ethic component.

Surveillance of Barrett's oesophagus allows us to study the evolutionary dynamics of a human neoplasm over time. Here we use multicolour fluorescence in situ hybridization on brush cytology specimens, from two time points with a median interval of 37 months in 195 non-dysplastic Barrett's patients, and a third time point in a subset of 90 patients at a median interval of 36 months, to study clonal evolution at single-cell resolution. Baseline genetic diversity predicts progression and remains in a stable dynamic equilibrium over time. Clonal expansions are rare, being detected once every 36.8 patient years, and growing at an average rate of 1.58 cm2 (95% CI: 0.09–4.06) per year, often involving the p16 locus. This suggests a lack of strong clonal selection in Barrett's and that the malignant potential of ‘benign' Barrett's lesions is predetermined, with important implications for surveillance programs., Barrett's oesophagus is thought to be a precursor lesion for oesophageal cancer, and predicting the benign lesions that progress to cancer is clinically important. Here, the authors use FISH to study the clonal evolution of Barrett's oesophagus and show that genetic diversity and somatic mutations are present early in the benign disease.

Objective The risk of developing adenocarcinoma in non-dysplastic Barrett9s oesophagus is low and difficult to predict. Accurate tools for risk stratification are needed to increase the efficiency of surveillance. We aimed to develop a prediction model for progression using clinical variables and genetic markers. Methods In a prospective cohort of patients with non-dysplastic Barrett9s oesophagus, we evaluated six molecular markers: p16 , p53 , Her-2/neu , 20q , MYC and aneusomy by DNA fluorescence in situ hybridisation on brush cytology specimens. Primary study outcomes were the development of high-grade dysplasia or oesophageal adenocarcinoma. The most predictive clinical variables and markers were determined using Cox proportional-hazards models, receiver operating characteristic curves and a leave-one-out analysis. Results A total of 428 patients participated (345 men; median age 60 years) with a cumulative follow-up of 2019 patient-years (median 45 months per patient). Of these patients, 22 progressed; nine developed high-grade dysplasia and 13 oesophageal adenocarcinoma. The clinical variables, age and circumferential Barrett9s length, and the markers, p16 loss, MYC gain and aneusomy, were significantly associated with progression on univariate analysis. We defined an ‘Abnormal Marker Count’ that counted abnormalities in p16, MYC and aneusomy, which significantly improved risk prediction beyond using just age and Barrett9s length. In multivariate analysis, these three factors identified a high-risk group with an 8.7-fold (95% CI 2.6 to 29.8) increased HR when compared with the low-risk group, with an area under the curve of 0.76 (95% CI 0.66 to 0.86). Conclusions A prediction model based on age, Barrett9s length and the markers p16, MYC and aneusomy determines progression risk in non-dysplastic Barrett9s oesophagus.

Extracellular cyclophilin A (CyPA) and CyPB have been well described as chemotactic factors for various leukocyte subsets, suggesting their contribution to inflammatory responses. Unlike CyPA, CyPB accumulates in extracellular matrixes, from which it is released by inflammatory proteases. Hence, we hypothesized that it could participate in tissue inflammation by regulating the activity of macrophages. In the current study, we confirmed that CyPB initiated in vitro migration of macrophages, but it did not induce production of proinflammatory cytokines. In contrast, pretreatment of macrophages with CyPB attenuated the expression of inflammatory mediators induced by LPS stimulation. The expression of TNF-α mRNA was strongly reduced after exposure to CyPB, but it was not accompanied by significant modification in LPS-induced activation of MAPK and NF-κB pathways. LPS activation of a reporter gene under the control of TNF-α gene promoter was also markedly decreased in cells treated with CyPB, suggesting a transcriptional mechanism of inhibition. Consistent with this hypothesis, we found that CyPB induced the expression of B cell lymphoma-3 (Bcl-3), which was accompanied by a decrease in the binding of NF-κB p65 to the TNF-α promoter. As expected, interfering with the expression of Bcl-3 restored cell responsiveness to LPS, thus confirming that CyPB acted by inhibiting initiation of TNF-α gene transcription. Finally, we found that CyPA was not efficient in attenuating the production of TNF-α from LPS-stimulated macrophages, which seemed to be due to a modest induction of Bcl-3 expression. Collectively, these findings suggest an unexpected role for CyPB in attenuation of the responses of proinflammatory macrophages.

Background Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. Methods To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. Results Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. Conclusions Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.)

Introduction: Inflammatory bowel disease (IBD) confers an increased lifetime risk of developing colorectal cancer (CRC). Our study aimed to compare the molecular and genetic features of colitis-associated CRC (CA-CRC) to the more common sporadic CRC (S-CRC), and to dissect the evolutionary history of CA-CRC using multi-region next generation sequencing. Methods: Fresh frozen colectomy specimens were collected from 12 patients with CA-CRC. For each case, whole exome sequencing was performed on multiple regions of tumor, adjacent normal mucosa and blood. Variants in key genes were validated by Sanger sequencing and BaseScope in situ hybridization, and copy numbers were validated by FISH. Copy number profiling (by SNP array or low-pass whole genome sequencing) was performed on low grade dysplasia (LGD; n=28), high grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=15). Phylogenetic trees were reconstructed for each case, and evolutionary analysis was used to reveal the temporal sequence of events leading to CA-CRC. Results: The majority of sequenced tumors (10/12) were microsatellite stable, and these had a median mutation rate of 3.0 single nucleotide alterations (SNAs) per Mb, ~20% higher than that of S-CRC. Mutational signatures identified ‘accelerated ageing' in the colitic bowel, likely a consequence of repeated inflammation and regeneration cycles. There was considerable mutational burden in non-dysplastic IBD mucosa (median 47 SNAs), with a median of 24% of these SNAs also clonal within the neighboring CA-CRC, indicating a mutational field. In CA-CRC the most commonly mutated gene was TP53, occurring more frequently than in S-CRC (80% vs. 58%), whereas APC mutations were significantly less common (40% vs. 75%, p=0.03). We analyzed the genetic heterogeneity of CA-CRCs and found that the number of clonal SNAs per tumor was not significantly different to S-CRCs, however CA-CRCs had significantly more SNAs that were unique to one region (p=0.04); this increase in diversity is likely due to an elevated mutation rate. CA- CRCs were often near-triploid (42%) or near-tetraploid (21%), with many other recurrent copy-number alterations that were distinct from those observed in S-CRC. Phylogenetic analysis revealed that copy number alterations (CNAs) accrue in non-dysplastic bowel, but the transition from LGD to HGD involves a punctuated ‘catastrophic' increase in CNA burden. Conclusions: Multi-region sequencing of CA-CRC has revealed a distinct pathway of colon carcinogenesis, with an increase in mutational burden and heterogeneity compared to S-CRC. Copy-number profiling indicated extensive genomic alterations, with dramatic accrual at the LGD to HGD transition. The significant mutational burden in surrounding normal mucosa indicates field cancerization, which is an encouraging prospect for screening programs; however the likelihood of punctuated evolution may offer a limited window for early detection. Citation Format: Ann-Marie Baker, William Cross, Kathleen Curtius, Chang-ho Ryan Choi, Ibrahim Al-Bakir, Daniel Temko, Pierre Martinez, Marc Williams, Hayley Davis, Sujata Biswas, Nicholas A. Wright, Morgan Moorghen, Stephen J. Hayes, Manuel Rodriguez-Justo, Andrew Silver, Lai Mun Wang, Marnix Jansen, Ailsa L. Hart, Simon J. Leedham, Trevor A. Graham. The evolutionary history of human colitis-associated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5368.

Heparan sulfate (HS) is recognized as an important player in a wide range of dynamic steps of inflammatory reactions. Thereby, structural HS remodeling is likely to play an important role in the regulation of inflammatory and immune responses; however, little is known about underlying mechanism. In this study, we analyzed the regulation of expression of HS 3-O-sulfotransferases (HS3STs) in response to inflammatory stimuli. We found that among the seven HS3ST isoenzymes, only the expression of HS3ST3B was markedly up-regulated in human primary monocytes and the related cell line THP1 after exposure to TLR agonists. TNF-α was also efficient, to a lesser extent, to increase HS3ST3B expression, while IL-6, IL-4, and IFN-γ were poor inducers. We then analyzed the molecular mechanisms that regulate the high expression of HS3ST3B in response to LPS. Based on the expression of HS3ST3B transcripts and on the response of a reporter gene containing the HS3ST3B1 promoter, we provide evidence that LPS induces a rapid and strong transcription of HS3ST3B1 gene, which was mainly dependent on the activation of NF-κB and JNK signaling pathways. Additionally, active p38 MAPK and de novo synthesized proteins are involved in post-transcriptional mechanisms to maintain a high level of HS3ST3B mRNA to a steady state. Altogether, our findings indicate that HS3ST3B1 gene behaves as a primary response gene, suggesting that it may play an important role in making 3-O-sulfated HS with specific functions in the regulation of inflammatory and immune responses. J. Cell. Biochem. 117: 1529-1542, 2016. © 2015 Wiley Periodicals, Inc.

Hepatitis C virus (HCV) entry involves binding to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction in the context of a viral infection. Patient sera and monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pulldown assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with the virion-heparin interaction, strongly suggesting that at the virion surface, HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pulldown experiments, and inhibition experiments with anti-apolipoprotein E antibodies indicated that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of the HS structural determinants required for HCV infection by silencing of the enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N - and 6- O -sulfation but not 2- O -sulfation is required for HCV infection and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures. IMPORTANCE Hepatitis C is a global health problem. Hepatitis C virus (HCV) infects approximately 130 million individuals worldwide, with the majority of cases remaining undiagnosed and untreated. In most infected individuals, the virus evades the immune system and establishes a chronic infection. As a consequence, hepatitis C is the leading cause of cirrhosis, end-stage liver disease, hepatocellular carcinoma, and liver transplantation. Virus infection is initiated by entry of the virus into the host cell. In this study, we provide new insights into the viral and cellular determinants involved in the first step of HCV entry, the binding of the virus to host cells. We show that apolipoprotein E is likely responsible for virus binding to heparan sulfate and that N - and 6- O -sulfation of the heparan sulfate proteoglycans is required for HCV infection. In addition, the minimal HS length unit required for HCV infection is a decasaccharide.

Macrophages are major cells of inflammatory process and take part in a large number of physiological and pathological processes. According to tissue environment, they can polarize into pro-inflammatory (M1) or alternative (M2) cells. Although many evidences have hinted to a potential role of cell-surface glycosaminoglycans (GAGs) in the functions of macrophages, the effect of M1 or M2 polarization on the biosynthesis of these polysaccharides has not been investigated so far. GAGs are composed of repeat sulfated disaccharide units. Heparan (HS) and chondroitin/dermatan sulfates (CS/DS) are the major GAGs expressed at the cell membrane. They are involved in numerous biological processes, which rely on their ability to selectively interact with a large panel of proteins. More than 20 genes encoding sulfotransferases have been implicated in HS and CS/DS biosynthesis, and the functional repertoire of HS and CS/DS has been related to the expression of these isoenzymes. In this study, we analyzed the expression of sulfotransferases as a response to macrophage polarization. We found that M1 and M2 activation drastically modified the profiles of expression of numerous HS and CS/DS sulfotransferases. This was accompanied by the expression of GAGs with distinct structural features. We then demonstrated that GAGs of M2 macrophages were efficient to present fibroblast growth factor-2 in an assay of tumor cell proliferation, thus indicating that changes in GAG structure may contribute to the functions of polarized macrophages. Altogether, our findings suggest a regulatory mechanism in which fine modifications in GAG biosynthesis may participate to the plasticity of macrophage functions.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Background Candidate biomarkers have been identified for clear cell renal cell carcinoma (ccRCC) patients, but most have not been validated. Objective To validate published ccRCC prognostic biomarkers in an independent patient cohort and to assess intratumour heterogeneity (ITH) of the most promising markers to guide biomarker optimisation. Design, setting, and participants Cancer-specific survival (CSS) for each of 28 identified genetic or transcriptomic biomarkers was assessed in 350 ccRCC patients. ITH was interrogated in a multiregion biopsy data set of 10 ccRCCs. Outcome measurements and statistical analysis Biomarker association with CSS was analysed by univariate and multivariate analyses. Results and limitations A total of 17 of 28 biomarkers (TP53 mutations; amplifications of chromosomes 8q, 12, 20q11.21q13.32, and 20 and deletions of 4p, 9p, 9p21.3p24.1, and 22q; low EDNRB and TSPAN7 expression and six gene expression signatures) were validated as predictors of poor CSS in univariate analysis. Tumour stage and the ccB expression signature were the only independent predictors in multivariate analysis. ITH of the ccB signature was identified in 8 of 10 tumours. Several genetic alterations that were significant in univariate analysis were enriched, and chromosomal instability indices were increased in samples expressing the ccB signature. The study may be underpowered to validate low-prevalence biomarkers. Conclusions The ccB signature was the only independent prognostic biomarker. Enrichment of multiple poor prognosis genetic alterations in ccB samples indicated that several events may be required to establish this aggressive phenotype, catalysed in some tumours by chromosomal instability. Multiregion assessment may improve the precision of this biomarker. Patient summary We evaluated the ability of published biomarkers to predict the survival of patients with clear cell kidney cancer in an independent patient cohort. Only one molecular test adds prognostic information to routine clinical assessments. This marker showed good and poor prognosis results within most individual cancers. Future biomarkers need to consider variation within tumours to improve accuracy., Take Home Message A total of 17 of 28 published biomarkers for clear cell renal cell carcinoma have been validated as predictors of survival. The ccA/ccB signature outperforms all others and adds prognostic information. Intratumour heterogeneity was seen for this biomarker, and multiregion assessment of tumours may further improve its accuracy.

PMCID: PMC4636053.-- et al., Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73-75% of identified ccRCC driver aberrations were subclonal confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development., C.S. and M. Gerlinger are supported by grants from Cancer Research UK Biomarkers and Imaging Discovery and Development Committee (BIDD), the Medical Research Council and the Seventh European Union Framework Programme, and C.S. is supported by the Breast Cancer Research Foundation and the Rosetrees Trust. We acknowledge the Ramón y Cajal program of the Ministerio de Economía y Competitividad, Spain, and Novartis for funding support for E-PREDICT clinical trials. This study was supported by researchers at the National Institute for Health Research Biomedical Research Centres at University College London Hospitals and at the Royal Marsden Hospital.

Despite recent progress thanks to next-generation sequencing technologies, personalised cancer medicine is still hampered by intra-tumour heterogeneity and drug resistance. As most patients with advanced metastatic disease face poor survival, there is need to improve early diagnosis. Analysing circulating tumour DNA (ctDNA) might represent a non-invasive method to detect mutations in patients, facilitating early detection. In this article, we define reduced gene panels from publicly available datasets as a first step to assess and optimise the potential of targeted ctDNA scans for early tumour detection. Dividing 4,467 samples into one discovery and two independent validation cohorts, we show that up to 76% of 10 cancer types harbour at least one mutation in a panel of only 25 genes, with high sensitivity across most tumour types. Our analyses demonstrate that targeting "hotspot" regions would introduce biases towards in-frame mutations and would compromise the reproducibility of tumour detection.

International audience; Distant metastasis account for about 90 % of cancer associated deaths, and yet the oncology field is cruelly lacking tools to accurately predict and/or prevent metastasis. Distant metastasis occurs when circulating tumor cells interact with the endothelium of distant organs and extravasate from the blood vessel into the surrounding tissue. Selectins are a family of carbohydrate receptors well depicted for their role in tumor cells extravasation. They mediate primary interactions of cancer cells with endothelial cells, as well as secondary interactions with leucocytes and platelets, which are also promoting metastasis. The cancer associated carbohydrate antigen sialyl-Lewis x (sLe(x)) has been repeatedly shown to be involved, as selectin ligand, in these interactions. However, recent studies have highlighted that glycosaminoglycans (GAGs), another class of glycans, may also serve as ligands for selectins. We report herein that cancer-associated GAGs are differentially recognized by selectins according to their density of sulfation and the pH conditions of the binding. We also show that these parameters regulate platelets-cancer cells heterotypic aggregation, supporting the idea that GAGs may have pro-metastatic function. Combining our experimental results with in depth analyses of molecular dockings, we propose a model of GAG/selectin interactions robust enough to recapitulate the differential binding of selectins to GAGs, the competition between GAGs and sLe(x) for selectin binding and the effect of sub-physiological pH on GAGs affinities towards selectins. Altogether, our data suggest GAGs to be good ligands for selectins, potentially promoting distant metastasis in a complementary way to sLe(x).

The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

Intratumour heterogeneity (ITH) may foster tumour adaptation and compromise the efficacy of personalized medicine approaches. The scale of heterogeneity within a tumour (intratumour heterogeneity) relative to genetic differences between tumours (intertumour heterogeneity) is unknown. To address this, we obtained 48 biopsies from eight stage III and IV clear cell renal cell carcinomas (ccRCCs) and used DNA copy-number analyses to compare biopsies from the same tumour with 440 single tumour biopsies from the Cancer Genome Atlas (TCGA). Unsupervised hierarchical clustering of TCGA and multi-region ccRCC samples revealed segregation of samples from the same tumour into unrelated clusters; 25% of multi-region samples appeared more similar to unrelated samples than to any other sample originating from the same tumour. We found that the majority of recurrent DNA copy number driver aberrations in single biopsies were not present ubiquitously in late-stage ccRCCs and were likely to represent subclonal events acquired during tumour progression. Such heterogeneous subclonal genetic alterations within individual tumours may impair the identification of robust ccRCC molecular subtypes classified by distinct copy number alterations and clinical outcomes. The co-existence of distinct subclonal copy number events in different regions of individual tumours reflects the diversification of individual ccRCCs through multiple evolutionary routes and may contribute to tumour sampling bias and impact upon tumour progression and clinical outcome.