Tissue specific patterns of methylated cytosine residues vary with age, can be altered by environmental factors, and are often abnormal in human disease yet the cellular consequences of DNA ...methylation are incompletely understood. Although the bodies of highly expressed genes are often extensively methylated in plants, the relationship between intragenic methylation and expression is less clear in mammalian cells. We performed genome-wide analyses of DNA methylation and gene expression to determine how the pattern of intragenic methylation correlates with transcription and to assess the relationship between methylation of exonic and intronic portions of the gene body. We found that dense exonic methylation is far more common than previously recognized or expected statistically, yet first exons are relatively spared compared to more downstream exons and introns. Dense methylation surrounding the transcription start site (TSS) is uncoupled from methylation within more downstream regions suggesting that there are at least two classes of intragenic methylation. Whereas methylation surrounding the TSS is tightly linked to transcriptional silencing, methylation of more downstream regions is unassociated with the magnitude of gene expression. Notably, we found that DNA methylation downstream of the TSS, in the region of the first exon, is much more tightly linked to transcriptional silencing than is methylation in the upstream promoter region. These data provide direct evidence that DNA methylation is interpreted dissimilarly in different regions of the gene body and suggest that first exon methylation blocks transcript initiation, or vice versa. Our data also show that once initiated, downstream methylation is not a significant impediment to polymerase extension. Thus, the consequences of most intragenic DNA methylation must extend beyond the modulation of transcription magnitude.Sequencing data and gene expression microarray data have been submitted to the GEO online database (accession number SRA012081.1). Supporting information including expanded methods and ten additional figures in support of the manuscript is provided.
The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is ...challenging, because clinically detectable inflammation and its treatment can alter the microbiota's composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-mediated colitis. Here we conduct a prospective study of patients with metastatic melanoma undergoing ipilimumab treatment and correlate the pre-inflammation faecal microbiota and microbiome composition with subsequent colitis development. We demonstrate that increased representation of bacteria belonging to the Bacteroidetes phylum is correlated with resistance to the development of checkpoint-blockade-induced colitis. Furthermore, a paucity of genetic pathways involved in polyamine transport and B vitamin biosynthesis is associated with an increased risk of colitis. Identification of these biomarkers may enable interventions to reduce the risk of inflammatory complications following cancer immunotherapy.
We integrated the genomic sequencing of 1,918 breast cancers, including 1,501 hormone receptor-positive tumors, with detailed clinical information and treatment outcomes. In 692 tumors previously ...exposed to hormonal therapy, we identified an increased number of alterations in genes involved in the mitogen-activated protein kinase (MAPK) pathway and in the estrogen receptor transcriptional machinery. Activating ERBB2 mutations and NF1 loss-of-function mutations were more than twice as common in endocrine resistant tumors. Alterations in other MAPK pathway genes (EGFR, KRAS, among others) and estrogen receptor transcriptional regulators (MYC, CTCF, FOXA1, and TBX3) were also enriched. Altogether, these alterations were present in 22% of tumors, mutually exclusive with ESR1 mutations, and associated with a shorter duration of response to subsequent hormonal therapies.
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•We performed prospective sequencing of 1,501 HR+ breast cancers in the clinical setting•MAPK and TF alterations were present in 22% of 692 HR+ post-endocrine therapy tumors•MAPK and TF alterations were mutually exclusive with ESR1 mutations•MAPK and TF alterations were associated with shorter response to endocrine therapies
Razavi et al. identify mutations in the MAPK pathway and the estrogen receptor transcriptional program in 22% of hormone receptor-positive breast cancers after hormone therapy. These mutations are mutually exclusive with ESR1 mutations and correlate with a shorter response duration to subsequent hormone therapies.
The intestinal microbiota contributes to the development of the immune system, and conversely, the immune system influences the composition of the microbiota. Toll-like receptors (TLRs) in the gut ...recognize bacterial ligands. Although TLR signaling represents a major arm of the innate immune system, the extent to which TLRs influence the composition of the intestinal microbiota remains unclear. We performed deep 16S ribosomal RNA sequencing to characterize the complex bacterial populations inhabiting the ileum and cecum of TLR- and MyD88-deficient mice. The microbiota of MyD88- and TLR-deficient mouse colonies differed markedly, with each colony harboring distinct and distinguishable bacterial populations in the small and large intestine. Comparison of MyD88-, TLR2-, TLR4-, TLR5-, and TLR9-deficient mice and their respective wild-type (WT) littermates demonstrated that the impact of TLR deficiency on the composition of the intestinal microbiota is minimal under homeostatic conditions and after recovery from antibiotic treatment. Thus, differences between TLR-deficient mouse colonies reflected long-term divergence of the microbiota after extended husbandry in isolation from each other. Long-term breeding of isolated mouse colonies results in changes of the intestinal microbiota that are communicated to offspring by maternal transmission, which account for marked compositional differences between WT and mutant mouse strains.
Highly diverse bacterial populations inhabit the gastrointestinal tract and modulate host inflammation and promote immune tolerance. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), ...the gastrointestinal mucosa is damaged, and colonizing bacteria are impacted, leading to an impaired intestinal microbiota with reduced diversity. We examined the impact of intestinal diversity on subsequent mortality outcomes following transplantation. Fecal specimens were collected from 80 recipients of allo-HSCT at the time of stem cell engraftment. Bacterial 16S rRNA gene sequences were characterized, and microbial diversity was estimated using the inverse Simpson index. Subjects were classified into high, intermediate, and low diversity groups and assessed for differences in outcomes. Mortality outcomes were significantly worse in patients with lower intestinal diversity; overall survival at 3 years was 36%, 60%, and 67% for low, intermediate, and high diversity groups, respectively (P = .019, log-rank test). Low diversity showed a strong effect on mortality after multivariate adjustment for other clinical predictors (transplant related mortality: adjusted hazard ratio, 5.25; P = .014). In conclusion, the diversity of the intestinal microbiota at engraftment is an independent predictor of mortality in allo-HSCT recipients. These results indicate that the intestinal microbiota may be an important factor in the success or failure in allo-HSCT.
•Intestinal diversity is predictive of mortality in allo-HSCT.
Metastatic colorectal cancers (mCRCs) are clinically heterogeneous, but the genomic basis of this variability remains poorly understood. We performed prospective targeted sequencing of 1,134 CRCs. We ...identified splice alterations in intronic regions of APC and large in-frame deletions in CTNNB1, increasing oncogenic WNT pathway alterations to 96% of CRCs. Right-sided primary site in microsatellite stable mCRC was associated with shorter survival, older age at diagnosis, increased mutations, and enrichment of oncogenic alterations in KRAS, BRAF, PIK3CA, AKT1, RNF43, and SMAD4 compared with left-sided primaries. Left-sided tumors frequently had no identifiable genetic alteration in mitogenic signaling, but exhibited higher mitogenic ligand expression. Our results suggest different pathways to tumorigenesis in right- and left-sided microsatellite stable CRC that may underlie clinical differences.
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•Prospective targeted sequencing of 1,134 colorectal cancers in the clinical setting•Few genomic differences between primaries and metastases•Identified CTNNB1 in-frame deletions enriched in microsatellite stable cases•Differences in APC, BRAF, KRAS, and NRAS predict varied survival by tumor laterality
Yaeger et al. perform prospective sequencing of metastatic colorectal cancers (mCRCs). Right-sided primary microsatellite stable mCRCs are associated with increased oncogenic mutations whereas most left-sided tumors lack identifiable genetic mitogenic signaling alterations but highly express mitogenic ligands.
Cancer spread to the central nervous system (CNS) often is diagnosed late and is unresponsive to therapy. Mechanisms of tumor dissemination and evolution within the CNS are largely unknown because of ...limited access to tumor tissue.
We sequenced 341 cancer-associated genes in cell-free DNA from cerebrospinal fluid (CSF) obtained through routine lumbar puncture in 53 patients with suspected or known CNS involvement by cancer.
We detected high-confidence somatic alterations in 63% (20 of 32) of patients with CNS metastases of solid tumors, 50% (six of 12) of patients with primary brain tumors, and 0% (zero of nine) of patients without CNS involvement by cancer. Several patients with tumor progression in the CNS during therapy with inhibitors of oncogenic kinases harbored mutations in the kinase target or kinase bypass pathways. In patients with glioma, the most common malignant primary brain tumor in adults, examination of cell-free DNA uncovered patterns of tumor evolution, including temozolomide-associated mutations.
The study shows that CSF harbors clinically relevant genomic alterations in patients with CNS cancers and should be considered for liquid biopsies to monitor tumor evolution in the CNS.
Background. Bacteremia is a frequent complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). It is unclear whether changes in the intestinal microbiota during allo-HSCT ...contribute to the development of bacteremia. We examined the microbiota of patients undergoing allo-HSCT, and correlated microbial shifts with the risk of bacteremia. Methods. Fecal specimens were collected longitudinally from 94 patients undergoing allo-HSCT, from before transplant until 35 days after transplant. The intestinal microbiota was characterized by 454 pyrosequencing of the V1-V3 region of bacterial 16S ribosomal RNA genes. Microbial diversity was estimated by grouping sequences into operational taxonomic units and calculating the Shannon diversity index. Phylogenetic classification was obtained using the Ribosomal Database Project classifier. Associations of the microbiota with clinical predictors and outcomes were evaluated. Results. During allo-HSCT, patients developed reduced diversity, with marked shifts in bacterial populations inhabiting the gut. Intestinal domination, defined as occupation of at least 30% of the microbiota by a single predominating bacterial taxon, occurred frequently. Commonly encountered dominating organisms included Enterococcus, Streptococcus, and various Proteobacteria. Enterococcal domination was increased 3-fold by metronidazole administration, whereas domination by Proteobacteria was reduced 10-fold by fluoroquinolone administration. As a predictor of outcomes, enterococcal domination increased the risk of Vancomycin-resistant Enterococcus bacteremia 9-fold, and proteobacterial domination increased the risk of gram-negative rod bacteremia 5-fold. Conclusions. During allo-HSCT, the diversity and stability of the intestinal flora are disrupted, resulting in domination by bacteria associated with subsequent bacteremia. Assessment of fecal microbiota identifies patients at highest risk for bloodstream infection during allo-HCST.
Androgen receptor (AR) splice variants lacking the ligand binding domain (ARVs), originally isolated from prostate cancer cell lines derived from a single patient, are detected in normal and ...malignant human prostate tissue, with the highest levels observed in late stage, castration-resistant prostate cancer. The most studied variant (called AR-V7 or AR3) activates AR reporter genes in the absence of ligand and therefore, could play a role in castration resistance. To explore the range of potential ARVs, we screened additional human and murine prostate cancer models using conventional and next generation sequencing technologies and detected several structurally diverse AR isoforms. Some, like AR-V7/AR3, display gain of function, whereas others have dominant interfering activity. We also find that ARV expression increases acutely in response to androgen withdrawal, is suppressed by testosterone, and in some models, is coupled to full-length AR (AR-FL) mRNA production. As expected, constitutively active, ligand-independent ARVs such as AR-V7/AR3 are sufficient to confer anchorage-independent (in vitro) and castration-resistant (in vivo) growth. Surprisingly, this growth is blocked by ligand binding domain-targeted antiandrogens, such as MDV3100, or by selective siRNA silencing of AR-FL, indicating that the growth-promoting effects of ARVs are mediated through AR-FL. These data indicate that the increase in ARV expression in castrate-resistant prostate cancer is an acute response to castration rather than clonal expansion of castration or antiandrogen-resistant cells expressing gain of function ARVs, and furthermore, they provide a strategy to overcome ARV function in the clinic.
Malignant peripheral nerve sheath tumors (MPNSTs) represent a group of highly aggressive soft-tissue sarcomas that may occur sporadically, in association with neurofibromatosis type I (NF1 ...associated) or after radiotherapy. Using comprehensive genomic approaches, we identified loss-of-function somatic alterations of the Polycomb repressive complex 2 (PRC2) components (EED or SUZ12) in 92% of sporadic, 70% of NF1-associated and 90% of radiotherapy-associated MPNSTs. MPNSTs with PRC2 loss showed complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3) and aberrant transcriptional activation of multiple PRC2-repressed homeobox master regulators and their regulated developmental pathways. Introduction of the lost PRC2 component in a PRC2-deficient MPNST cell line restored H3K27me3 levels and decreased cell growth. Additionally, we identified frequent somatic alterations of CDKN2A (81% of all MPNSTs) and NF1 (72% of non-NF1-associated MPNSTs), both of which significantly co-occur with PRC2 alterations. The highly recurrent and specific inactivation of PRC2 components, NF1 and CDKN2A highlights their critical and potentially cooperative roles in MPNST pathogenesis.
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