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© 2014 Nature America, Inc. All rights reserved., Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA., This work was supported in part by an IDEAS grant from Princess Margaret Hospital, the Head and Neck Translational Research Program (I.W., B.A.C., P.C.B. and J.D.M.), the Ontario Institute for Cancer Research and the government of Ontario (P.C.B. and J.D.M.) and by a Terry Fox Research Institute New Investigator Award (P.C.B.). C.H. and F.-F.L. acknowledge support from the Wharton family, Joe's Team, Gordon Tozer, the Campbell Family Institute for Cancer Research and the Ministry of Health and Long-Term Planning, Canada.

Aims Current heart failure (HF) guidelines highlight the importance of iron deficiency (ID) in HF. Whether HF itself or age-related comorbidities contribute to ID is uncertain, and previous data were limited to Western populations. We aimed to study the prevalence, clinical correlates, functional significance and prognosis of ID in HF patients, compared with community-based controls in a multi-ethnic Southeast Asian population. Methods and results Iron status was assessed in 751 HF patients (age 62.0 ± 12.2 years, 75.5% men, 64.7% Chinese, 23.9% Malay, 10.2% Indian) and 601 controls (age 56.9 ± 10.4 years, 49.8% men, 70.9% Chinese, 21.5% Malay, 7.2% Indian). ID, defined as ferritin

In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell-type-specific regulation directed by DNA binding factors. This comparatively static landscape is in marked contrast with the events of fertilization, during which the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, although methylation remains detectable at several notable features. These dynamics have been extensively characterized in the mouse, with only limited observations available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of residual maintenance are primarily restricted to gene bodies. Although most features share similar dynamics to those in mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island promoters that extend beyond known imprint control regions. Retrotransposon regulation is also highly diverse, and transitions from maternally to embryonically expressed elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation., National Institutes of Health (U.S.) (Pioneer Award 5DP1OD003958), Burroughs Wellcome Fund (Career Award at the Scientific Interface), National Human Genome Research Institute (U.S.). Centers of Excellence in Genomic Science (1P50HG006193-01)

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways.

Non-proteolytic ubiquitin signaling mediated by Lys63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells.

e15120 Background: Assays detecting methylated circulating tumor DNA (ctDNA) hold promise for colorectal cancer (CRC) screening, but there is a need to develop more accurate assays for early stages. In this study, we examined modifications to COLVERA (an IKZF1 and BCAT1 methylation marker qPCR assay) that added a third biomarker (IRF4) as well as introducing double stranded detection. Methods: Bisulfite converted DNA extracted from plasma collected from subjects undergoing diagnosis for CRC was assayed using either a double-stranded (ds) specific multiplexed qPCR for BCAT1 and IKZF1 detection (Assay 1) and/or a single-stranded (ss) specific BCAT1/IRF4, ds-specific IKZF1 multiplexed qPCR (Assay 2). The unmodified COLVERA test (Clinical Genomics) was used for comparison. Detection of any marker was considered positive. Results: Assay 1 performance evaluated against COLVERA on bisulfite converted DNA from 1453 samples showed an improved sensitivity for CRC detection (93/134 (69.4%) vs 79/134 (59.0%) COLVERA, p = 0.02), but specificity was suboptimal (no neoplasia: 861/1023 (84.2%) vs 946/1023 (92.5%) COLVERA, p < 0.0001). The decrease in specificity resulted primarily from modifications in the BCAT1 assay component (1023 no neoplasia: 60 positive with ss- BCAT1 vs 146 positive with ds- BCAT1, p < 0.0001). Thus, a novel configuration, Assay 2 (ss- BCAT1, ss- IRF4 and ds- IKZF1) was compared to Assay 1 using 189 previously untested specimens. Assay 2 showed a notable improvement in specificity (99/104 (95.2%) vs 93/104 (89.4%) Assay 1; p = 0.0351). Both assays exhibited comparable sensitivities for CRC (41/49 (83.7%) vs 39/49 (79.6%), Assay 1, p = 0.3633). Assay 2 was positive in: adenomas, 9/36 (25.0%); Stage I, 3/7 (42.9%); Stage II, 17/19 (89.5%); Stage III, 6/6 (100%); Stage IV, 7/7 (100%) and 8/10 (80%) unstaged CRCs. Prior COLVERA sensitivity estimates were 9% adenoma, 41% Stage I and 76% Stage II. Conclusions: Targeting both of the non-complementary bisulfite converted strands of selective regions of interest markedly improve ctDNA assay sensitivity for cancer including early lesions while achieving good specificity. Prospective evaluation in a true CRC screening population is now underway. Clinical trial information: 12611000318987.

We integrated clinical, genomic, and transcriptomic data from 224 primaries and 95 metastases from 289 patients to characterize progression of pancreatic ductal adenocarcinoma (PDAC). Driver gene alterations and mutational and expression-based signatures were preserved, with truncations, inversions, and translocations most conserved. Cell cycle progression (CCP) increased with sequential inactivation of tumor suppressors, yet remained higher in metastases, perhaps driven by cell cycle regulatory gene variants. Half of the cases were hypoxic by expression markers, overlapping with molecular subtypes. Paired tumor heterogeneity showed cancer cell migration by Halstedian progression. Multiple PDACs arising synchronously and metachronously in the same pancreas were actually intra-parenchymal metastases, not independent primary tumors. Established clinical co-variates dominated survival analyses, although CCP and hypoxia may inform clinical practice.

The motility of bacteria plays a key role in their colonization of surfaces during infection. Derivatives of cranberry fruit have been shown to interfere with bacterial motility. Herein, we report on the incorporation of cranberry derived materials (CDMs) into silicone substrates with the aim of impairing bacterial pathogen motility and spreading on the substrate surface. The release of CDMs from the silicone substrates when soaking in an aqueous medium was quantified for a period of 24h. Next, we showed that CDMs released from two silicone substrates remain bioactive as they downregulate the expression of the flagellin gene of two key uropathogens - Escherichia coli CFT073 and Proteus mirabilis HI4320. Furthermore, we demonstrate that CDM-modified silicone inhibits the swarming motility of P. mirabilis, an aggressive swarmer. The bioactive, CDM-modified substrates can find broad applications in the medical device and food industries where the impairment of bacterial colonization of surfaces is of paramount importance.

Understanding how patients with heart failure with preserved ejection fraction (HFPEF) die provides insight into the natural history and pathophysiology of this complex syndrome, thereby allowing better prediction of response to therapy in designing clinical trials. This review summarizes the current state of knowledge surrounding mortality rates, modes of death, and prognostic factors in HFPEF. Despite the lack of uniform reporting, the following conclusions may be drawn from previous studies. The mortality burden of HFPEF is substantial, ranging from 10% to 30% annually, and higher in epidemiological studies than in clinical trials. Mortality rates compared with heart failure with reduced ejection fraction (HFREF) appear to be strongly influenced by the type of study, but are clearly elevated compared with age- and co-morbidity-matched controls without heart failure. The majority of deaths in HFPEF are cardiovascular deaths, comprising 51-60% of deaths in epidemiological studies and ∼70% in clinical trials. Among cardiovascular deaths, sudden death and heart failure death are the leading cardiac modes of death in HFPEF clinical trials. Compared with HFREF, the proportions of cardiovascular deaths, sudden death, and heart failure deaths are lower in HFPEF. Conversely, non-cardiovascular deaths constitute a higher proportion of deaths in HFPEF than in HFREF, particularly in epidemiological studies, where this difference may be related to fewer coronary heart deaths in HFPEF. Key mortality risk factors, including age, gender, body mass index, burden of co-morbidities, and coronary artery disease, offer some explanation for the differences in mortality rates observed across studies.

We describe a novel graphical representation of a crystallization condition that provides an intuitive guide to setting the upper and lower concentration boundaries in a fine screen based around that condition.

Background. Next-generation sequencing of cancers has identified important therapeutic targets and biomarkers. The goal of this pilot study was to compare the genetic changes in a human papillomavirus- (HPV-)positive and an HPV-negative head and neck tumor. Methods. DNA was extracted from the blood and primary tumor of a patient with an HPV-positive tonsillar cancer and those of a patient with an HPV-negative oral tongue tumor. Exome enrichment was performed using the Agilent SureSelect All Exon Kit, followed by sequencing on the ABI SOLiD platform. Results. Exome sequencing revealed slightly more mutations in the HPV-negative tumor (73) in contrast to the HPV-positive tumor (58). Multiple mutations were noted in zinc finger genes (ZNF3, 10, 229, 470, 543, 616, 664, 638, 716, and 799) and mucin genes (MUC4, 6, 12, and 16). Mutations were noted in MUC12 in both tumors. Conclusions. HPV-positive HNSCC is distinct from HPV-negative disease in terms of evidence of viral infection, p16 status, and frequency of mutations. Next-generation sequencing has the potential to identify novel therapeutic targets and biomarkers in HNSCC.

Importance Outcomes for patients with pancreatic ductal adenocarcinoma (PDAC) remain poor. Advances in next-generation sequencing provide a route to therapeutic approaches, and integrating DNA and RNA analysis with clinicopathologic data may be a crucial step toward personalized treatment strategies for this disease. Objective To classify PDAC according to distinct mutational processes, and explore their clinical significance. Design, Setting, and Participants We performed a retrospective cohort study of resected PDAC, using cases collected between 2008 and 2015 as part of the International Cancer Genome Consortium. The discovery cohort comprised 160 PDAC cases from 154 patients (148 primary; 12 metastases) that underwent tumor enrichment prior to whole-genome and RNA sequencing. The replication cohort comprised 95 primary PDAC cases that underwent whole-genome sequencing and expression microarray on bulk biospecimens. Main Outcomes and Measures Somatic mutations accumulate from sequence-specific processes creating signatures detectable by DNA sequencing. Using nonnegative matrix factorization, we measured the contribution of each signature to carcinogenesis, and used hierarchical clustering to subtype each cohort. We examined expression of antitumor immunity genes across subtypes to uncover biomarkers predictive of response to systemic therapies. Results The discovery cohort was 53% male (n = 79) and had a median age of 67 (interquartile range, 58-74) years. The replication cohort was 50% male (n = 48) and had a median age of 68 (interquartile range, 60-75) years. Five predominant mutational subtypes were identified that clustered PDAC into 4 major subtypes: age related, double-strand break repair, mismatch repair, and 1 with unknown etiology (signature 8). These were replicated and validated. Signatures were faithfully propagated from primaries to matched metastases, implying their stability during carcinogenesis. Twelve of 27 (45%) double-strand break repair cases lacked germline or somatic events in canonical homologous recombination genes—BRCA1,BRCA2,orPALB2. Double-strand break repair and mismatch repair subtypes were associated with increased expression of antitumor immunity, including activation of CD8-positive T lymphocytes (GZMAandPRF1) and overexpression of regulatory molecules (cytotoxic T-lymphocyte antigen 4, programmed cell death 1,and indolamine 2,3-dioxygenase 1), corresponding to higher frequency of somatic mutations and tumor-specific neoantigens. Conclusions and Relevance Signature-based subtyping may guide personalized therapy of PDAC in the context of biomarker-driven prospective trials.

Background A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. Results We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. Conclusions In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0090-4) contains supplementary material, which is available to authorized users.

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus–host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus–host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)--including many CpG island promoters--that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.

In humans, uropathogenic Escherichia coli (UPEC) is the most common etiological agent of uncomplicated urinary tract infections (UTIs). Cranberry extracts have been linked to the prevention of UTIs for over a century; however, a mechanistic understanding of the way in which cranberry derivatives prevent bacterial infection is still lacking. In this study, we used a fliC-lux reporter as well as quantitative reverse transcription-PCR to demonstrate that when UPEC strain CFT073 was grown or exposed to dehydrated, crushed cranberries or to purified cranberry-derived proanthocyanidins (cPACs), expression of the flagellin gene ( fliC ) was inhibited. In agreement with these results, transmission electron microscopy imaging of bacteria grown in the presence of cranberry materials revealed fewer flagella than those in bacteria grown under control conditions. Furthermore, we showed that swimming and swarming motilities were hindered when bacteria were grown in the presence of the cranberry compounds. Because flagellum-mediated motility has been suggested to enable UPEC to disseminate to the upper urinary tract, we propose that inhibition of flagellum-mediated motility might be a key mechanism by which cPACs prevent UTIs. This is the first report to show that cranberry compounds inhibit UPEC motility via downregulation of the fliC gene. Further studies are required to establish whether these inhibitors play a role in vivo .

Pathological lesions in and around a joint can arise from underlying dermis, subcutis, deep muscle, bone or synovium. Clinical presentation can include joint pain, joint swelling, palpable masses and mechanical restriction. Whilst giant cell tumour of tendon sheath, pigmented villonodular synovitis, synovial chondromatosis, lipoma arborescens, juxta articular myxomas and inflammatory arthritis are the better-known conditions of the joint. Intra-articular nodular fasciitis, on the other hand, is less well recognized both clinically and radiologically. It is rarely seen in routine practice and is only described in case reports in the literature. Due to the non-specific clinical and radiological findings as well as the unfamiliarity with the entity, the diagnosis of intra-articular nodular fasciitis is usually clinched only after histological examination. We present a case of intra-articular nodular fasciitis arising in the knee joint which was not suspected clinically or radiologically. Key Words Intra-articular, nodular fasciitis, joint, knee, MRI

Disease recurrence remains a significant cause of mortality in B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples have demonstrated that relapse arises from a minor subclone already present at diagnosis and not the dominant clone in the majority of patients. However, the reasons why only some clones survive therapy and generate relapse are obscure and elucidation of the mechanisms that underlie these differing fates may be revealed by functional analysis of isolated subclones. Previous work has shown that the subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients with varying cytogenetic abnormalities. Diagnosis-specific, relapse-specific, and shared clonal and subclonal variants were identified by whole exome sequencing of the patient samples. Targeted sequencing of these variants in 372 xenografts generated by transplantation of CD19+ cells in a limiting cell dilution assay uncovered clonal variation. This analysis provided for the unequivocal identification of minor subclones ancestral to the relapse, termed diagnosis Relapse-Initiating (dRI) clones, in the diagnostic sample. Our xenografting approach enabled the physical isolation of dRI clones providing a unique opportunity to interrogate their epigenetic and transcriptional landscapes in order to unravel their relapse initiating capacity. To this end, representative diagnosis, dRI and relapse clones from 5 of the 14 patients were subjected to RNAseq and ATACseq (assay for transposase-accessible chromatin using sequencing) analysis. Despite the differences in transcriptional and chromatin openness between patients, principal component analysis of subclones from individual patients positioned the dRI clones as evolutionary intermediates between the diagnosis and relapse clones. Hierarchical clustering of the most significantly differentially expressed genes and open chromatin regions demonstrated that dRI clones shared gene expression and chromatin accessibility signatures with both the dominant diagnosis clone as well as the dominant relapse clone. To gain mechanistic insight into the data we used gene set enrichment analysis (GSEA) and identified common molecular pathways present in all patients that were enriched in dRI clones and persisted at the time of relapse in comparison to the dominant diagnosis clone. dRI and relapse clones converged in the activation of genes involved in cellular functions such as endocytosis, autophagy and innate immune response. In addition, cell surface proteins like ABC transporters and ephrins were also upregulated in dRI and relapse clones. Remarkably, functional interrogation of dRI clones in secondary xenografts, in comparison to more representative diagnosis clones, displayed increased tolerance to standard chemotherapeutic agents (dexamethasone, L-asparaginase and vincristine). Investigation of the molecular pathways and cellular receptors/transporters identified by gene expression analysis are being assessed in vitro and in vivo as potential targets for novel therapeutic approaches and disease monitoring. Overall, we have shown evidence that minor subclones at diagnosis, ancestral to the relapse clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. In depth analysis of pathways identified in these dRI subclones will shed light on potential new therapeutic approaches for abrogating and reducing disease recurrence in B-ALL. Disclosures Mullighan: Amgen: Honoraria, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding.

Background Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ ALL), the most common form of ALL in adults, is a highly aggressive blood malignancy defined by the BCR-ABL1 fusion. Although inhibitors targeting the BCR-ABL1 oncoprotein, such as imatinib, have significantly improved clinical response rates for this disease, a subset of patients are refractory to therapy or respond initially but relapse soon after. ABL1 kinase domain mutations partly explain differential responses in patients; however, for the majority of cases, a molecular basis that can reconcile this clinical observation is lacking. Methods Flow-sorted blasts from 53 primary samples, 49 de novo Ph+ ALL and 4 lymphoid blast crisis CML, were subjected to RNA sequencing (RNA-seq) and whole genome sequencing (WGS). Response rates were tracked using BCR-ABL1 transcript levels from patient blood. Results Hierarchical clustering of transcriptome data produced two molecular subgroups of Ph+ ALL. One subgroup, which we termed 'Core-B', upregulated key regulators of B-lymphoid differentiation including IL7R and MS4A1 (CD20). By contrast, the second subgroup upregulated an expression program related to hematopoietic stem cell (HSC) and myeloid differentiation, with upregulation of KIT, CD34, MPO, CSF3R, and GATA3. We termed this subgroup 'Aberrant-Stem-Myeloid' (ASM). These subgroups displayed a striking disparity in response rates to intensive chemotherapy with imatinib. Whereas 'Core-B' patients showed highly durable responses often lasting many years, 'ASM' patients frequently relapsed (4% vs 43% relapse; p=0.007). We used WGS analysis to investigate the genetic basis of these molecular subtypes. 'Core-B' Ph+ ALLs were enriched for deletions in PAX5, a B-cell specification gene, and CDKN2A/B, tumor suppressors. The 'ASM' subtype lacked these genetic alterations; instead, these leukemias were enriched for deletions in EBF1, an early B-cell lineage factor that represses T-lymphoid and myeloid lineages and is expressed before PAX5 in B-cell lineage differentiation. Accordingly, blasts from 'ASM' leukemias with EBF1 deletions showed decreased CD19 antigen expression and upregulation of myeloid antigens by clinical flow cytometry. Rare cases with concurrent EBF1 and PAX5 deletions showed expression features of both 'ASM' and 'Core-B' leukemias. Mutations observed in myeloid leukemias (TET2, RUNX1) were only present in the 'ASM' subtype. Loss of IKZF1, found in 77% of cases, also displayed distinct patterns between the two subgroups; deletions leading to the dominant negative isoform (Ik6) were enriched in the 'Core-B' subgroup (45% vs. 14%; p=0.019) while monosomy 7 and large deletions encompassing IKZF1 were enriched in the 'ASM' leukemias (41% vs. 10%; p=0.017). In 1 of 4 diagnosis/relapse patients analyzed, a molecular switch from 'Core-B' at diagnosis to 'ASM' at relapse was observed. The diagnostic 'Core-B' clone from this patient harbored a PAX5 mutation that was lost at relapse, whereas the relapsed 'ASM' clone harbored trisomy 21 and a RUNX1 mutation. Altogether, our data suggest that the 'ASM' leukemias emerge through dysregulation of genes earlier in lympho-myeloid specification compared to 'Core-B' leukemias. These findings led us to investigate if the 'ASM' subtype originates from an HSC and the 'Core-B' subtype originates from a B-cell progenitor. We first looked at the distribution of the long (p210) and short (p190) isoforms of BCR-ABL1 in the two subtypes. The p210 isoform, also the hallmark of CML, is speculated to arise in an HSC, and the p190 is thought to arise in a B-cell progenitor. Neither the p190 or p210 BCR-ABL1 isoform was enriched in either subgroup. We resolved highly purified HSC and progenitor subsets from CD34+CD19- cells, functionally evaluated by methylcellulose assays, and subjected them to a sensitive nested-PCR strategy. Cases from both the 'ASM' and 'Core-B' subtypes showed HSC/myeloid progenitor involvement regardless of the BCR-ABL1 isoform. This data suggest that the cell-of-origin does not play a role in establishing the molecular subtype of leukemia blasts. Conclusion There are two distinct molecular subtypes of Ph+ ALL that demonstrate differential responses to treatment and emerge from independent mutational routes. Moreover, the key genetic determinants that form the molecular subtype are secondary driver alterations that lie downstream of BCR-ABL1. Disclosures No relevant conflicts of interest to declare.

In myeloproliferative neoplasms (MPN), somatic mutations in recurrently mutated myeloid leukemia genes, apart from MPN driver genes, are common and can increase transformation risk to acute myeloid leukemia (AML). However, the temporal acquisition of these mutations, the properties of the cells bearing these mutations, and how each mutation cooperates to promote leukemic transformation remains largely unknown. We therefore examined blood and bone marrow samples collected serially from patients with myelofibrosis (MF) who developed AML to further elucidate the genetic basis of leukemic transformation. Specific aims include: 1) determining the timeline of acquisition of mutations that contribute to AML in chronic phase and 2) defining the cell type that harbours the clone fated to initiate AML. Ten patients who transformed to AML following a diagnosis of MF (6 PMF, 3 post-PV MF, 1 post-ET MF) were studied. The time interval from the first MPN collection to AML diagnosis ranged between 1.5 to 6.6 years; and in 4 patients additional MPN samples collected between these time points were available for study. Somatic mutations were identified from whole genome sequencing (WGS) data of AML blasts (CD45dim cells), the MPN clone (CD45highCD33+ and CD34+ cells), and germline references (T-cells or Buccal DNA); and from targeted sequencing results of 54 recurrently mutated myeloid leukemia genes performed on AML samples as part of another study. Stem and progenitor populations (HSC, MPP, LMPP, CMP, MEP, GMP), as well as, mature cell populations (myeloid, erythroid, T-lymphoid, B-lymphoid and NK) were sorted from MPN time points and whole genome amplified using the REPLI-g Single Cell Kit (Qiagen). Somatic mutations were tracked by droplet digital PCR (ddPCR) and/or targeted sequencing. We identified an average of 5 (range 1-8) recurrently mutated myeloid leukemia genes that were somatically mutated in each patient at AML diagnosis, with an average of 5.5 (range 1-11) different mutations affecting these genes. Many of these mutations were present at ≥5% variant allele frequency (VAF) in the MPN clone at the earliest chronic phase time point. In 8 patients, the MPN driver mutation was detected in the MPN clone and AML blasts (MPNdriver-concordant), while in 2 other patients there was discordance as JAK2V617F was present in the MPN clone, but not the AML blasts. Myeloid leukemia genes that were mutated in 2 or more patients included SRSF2 (n=5), ASXL1 (n=4), TET2 (n=4), IDH1/2 (n=4), RUNX1 (n=4), NRAS (n=4), KRAS (n=2), U2AF1 (n=2), PHF6 (n=2), and STAG2 (n=2). We tracked mutations by ddPCR in populations sorted from MPN time points and demonstrated that 10 out of 13 mutations not identified by WGS were in fact present at low VAF in one or more cell populations. Moreover, in 5 patients with MPNdriver-concordant AML, the AML-fated clone was detectable 1.5 to 3 years before AML was diagnosed; and in 4 of these patients the AML-fated clone resided among cells with a HSC or MPP immunophenotype. In order to better establish the reservoir harbouring AML-fated clones, clonal tracking within the stem and progenitor populations by targeted sequencing of a greater number of AML-specific variants is underway. Among the two patients with JAK2V617F-negative AML, we observed in one patient the emergence of the AML-fated clone within one year of AML diagnosis. In summary, we show that clones fated to intiate AML in patients with MF often reside among cells with a HSC or MPP immunophenotype and can appear several years prior to the diagnosis of AML. This strongly suggests that mechanisms beyond the acquisition of somatic driver mutations (including but not limited to epigenetic alterations, acquisition of non-coding mutations, inflammation) are necessary to effectively promote full leukemic transformation. Interestingly, in one patient with JAK2V617F-negative AML (following JAK2V617F-positive MF), the latency period between the appearance of the AML-fated clone and AML diagnosis was much shorter, suggesting that the mechanisms underlying transformation are likely different for these 2 clonal evolutionary pathways. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding.

Memory often requires knowledge of the order of events. Previous findings about immediate judgments of relative order in short, subspan lists are variable regarding whether participants' strategy is to search memory in the forward direction, starting from the first list item and progressing toward the end item, or in the backward direction, starting from the end item and progressing toward the start. We asked whether wording of the instructions influences participants' search direction. Participants studied sequences of three to six consonants, and for an immediate, two-item probe of each list, judged which probe was presented earlier ("earlier" instruction) or later ("later" instruction) on the list. Forward and backward searches were supported for "earlier" and "later" instructions, respectively. Our findings suggest that participants have more than one effective strategy for order judgments in short lists, and that subtle instructional differences can bias memory search in either the forward or backward direction.

Introduction Tandem mass spectrometry (MS/MS) has emerged as a cornerstone of proteomic screens aimed at discovering putative protein biomarkers of disease with potential clinical applications. Systematic validation of lead candidates in large numbers of samples from patient cohorts remains an important challenge. One particularly promising high throughout technique is multiple reaction monitoring (MRM), a targeted form of MS/MS by which precise peptide precursor–product ion combinations, or transitions, are selectively tracked as informative probes. Despite recent progress, however, many important computational and statistical issues remain unresolved. These include the selection of an optimal set of transitions so as to achieve sufficiently high specificity and sensitivity when profiling complex biological specimens, and the corresponding generation of a suitable scoring function to reliably confirm tentative molecular identities based on noisy spectra. Methods In this study, we investigate various empirical criteria that are helpful to consider when developing and interpreting MRM-style assays based on the similarity between experimental and annotated reference spectra. We also rigorously evaluate and compare the performance of conventional spectral similarity measures, based on only a few pre-selected representative transitions, with a generic scoring metric, termed T corr, wherein a selected product ion profile is used to score spectral comparisons. Conclusions Our analyses demonstrate that T corr is potentially more suitable and effective for detecting biomarkers in complex biological mixtures than more traditional spectral library searches.

Abstract Many protocols have been used in clinical and research settings for collecting systolic blood pressure (SBP) measurements to calculate the ankle–brachial index (ABI); however, it is not known how useful it is to replicate measurements and which measures best reflect cardiovascular risk. Standardized measurements of ankle and arm SBP from 5140 overweight or obese individuals with type 2 diabetes were used to estimate sources of variation. Measurement characteristics of leg-specific ABI, as calculated using a standard algorithm based on the highest SBP of the dorsalis pedis or posterior tibial arteries, were projected using simulations. Coefficients of variability ranged from 2% to 3% when single SBP measurements were used and ABI was overestimated by 2–3%. Taking two SBP measurements at each site reduced standard errors and bias each by 30–40%. The sensitivity of detecting low ABI ranges exceeded 90% for ABI within 0.05 of the 0.90 clinical cut-point. The average and the minimum of the two (i.e. right and left) leg-specific ABI values had similar U-shaped relationships with Framingham risk scores; however, the average leg ABI had slightly greater precision. Replicating SBP measurements reduces the error and bias of ABI. Averaging leg-specific values may increase power for characterizing cardiovascular disease risk.

China has been criticized for using ‘no strings attached’ investment and aid packages to secure access to natural resources in Africa. Are these concerns legitimate or simply defensive reactions as China's rising influence in Africa challenges western hegemony? Examples of investment-for-resources swaps in four countries are examined, and the authors find that nature and relative development benefits of such deals cannot be generalized. Development (2007) 50, 63–68. doi:10.1057/palgrave.development.1100403

Organisms must carefully regulate energy intake and expenditure to balance growth and trade-offs with other physiological processes. This regulation is influenced by key pathways controlling appetite, feeding behaviour and energy homeostasis. Growth hormone (GH) transgenesis provides a model where food intake can be elevated, and is associated with dramatic modifications of growth, metabolism, and feeding behaviour, particularly in fish. RNA-Seq and qPCR analyses were used to compare the expression of multiple genes important in appetite regulation within brain regions and the pituitary gland (PIT) of GH transgenic (fed fully to satiation or restricted to a wild-type ration throughout their lifetime) and wild-type coho salmon (Oncorhynchus kisutch). RNA-Seq results showed that differences in both genotype and ration levels resulted in differentially expressed genes associated with appetite regulation in transgenic fish, including elevated Agrp1 in hypothalamus (HYP) and reduced Mch in PIT. Altered mRNA levels for Agrp1, Npy, Gh, Ghr, Igf1, Mch and Pomc were also assessed using qPCR analysis. Levels of mRNA for Agrp1, Gh, and Ghr were higher in transgenic than wild-type fish in HYP and in the preoptic area (POA), with Agrp1 more than 7-fold higher in POA and 12-fold higher in HYP of transgenic salmon compared to wild-type fish. These data are consistent with the known roles of orexigenic factors on foraging behaviour acting via GH and through MC4R receptor-mediated signalling. Igf1 mRNA was elevated in fully-fed transgenic fish in HYP and POA, but not in ration-restricted fish, yet both of these types of transgenic animals have very pronounced feeding behaviour relative to wild-type fish, suggesting IGF1 is not playing a direct role in appetite stimulation acting via paracrine or autocrine mechanisms. The present findings provide new insights on mechanisms ruling altered appetite regulation in response to chronically elevated GH, and on potential pathways by which elevated feeding response is controlled, independently of food availability and growth.

G-protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of different metabolic organs. First identified as part of the N-CoR corepressor complex, GPS2 has been...

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Enucleated oocytes have the distinctive ability to reprogram somatic nuclei back to totipotency. Here, we investigate genome-scale DNA methylation patterns after nuclear transfer and compare them to the dynamics at fertilization. We identify specific targets for DNA demethylation after nuclear transfer, such as germline-associated promoters, as well as unique limitations that include certain repetitive element classes.

Background: The use of inhaled corticosteroid (ICS) medications has been shown to improve asthma control and reduce asthma-related morbidity and mortality. Two recent randomized trials demonstrated dramatic improvements in ICS adherence by monitoring adherence with electronic sensors and providing automated reminders to participants to take their ICS medications. Given their lower levels of adherence and higher levels of asthma-related emergency department (ED) visits, hospitalizations, and death, urban minority populations could potentially benefit greatly from these types of interventions. Objective: The principal objective of this study will be to evaluate the feasibility, acceptability, and limited efficacy of a text message (short message service, SMS) reminder intervention to enhance ICS adherence in an urban minority population of children with asthma. We will also assess trajectories of ICS adherence in the 2 months following asthma hospitalization. Methods: Participants will include 40 children aged 2-13 years, who are currently admitted to the Children’s Hospital of Philadelphia (CHOP) for asthma, and their parent or legal guardian. Participants will be assigned to intervention and control arms using a 1:1 randomization scheme. The intervention arm will receive daily text message reminders for a 30-day intervention phase following hospitalization. This will be followed by a 30-day follow-up phase, in which all participants may choose whether or not to receive the text messages. Feasibility will be assessed by measuring (1) retention of the participants through the study phases and (2) perceived usefulness, acceptability, and preferences regarding the intervention components. Limited efficacy outcomes will include percent adherence to prescribed ICS regimen measured using Propeller Health sensors and change in parent-reported asthma control. We will perform an exploratory analysis to assess for discrete trajectories of adherence using group-based trajectory modeling (GBTM). Results: Study enrollment began in December 2015 and the intervention and follow-up phases are ongoing. Results of the data analysis are expected to be available by December 2016. Conclusions: This study will add to the literature by providing foundational feasibility data on which elements of a mobile health text-message reminder intervention may need to be modified to suit the needs and constraints of high-risk urban minority populations. Trial Registration: Clinicaltrials.gov NCT02615743; https://www.clinicaltrials.gov/ct2/show/study/NCT02615743 (Archived with WebCite at http://www.webcitation.org/6ji59rAXN) [JMIR Res Protoc 2017;6(3):e48]

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

The judgement of relative order (JOR) procedure is used to investigate serial-order memory. Measuring response times, the wording of the instructions (whether the earlier or the later item was designated as the target) reversed the direction of search in subspan lists (Chan, Ross, Earle, & Caplan Psychonomic Bulletin & Review, 16(5), 945–951, 2009). If a similar congruity effect applied to above-span lists and, furthermore, with error rate as the measure, this could suggest how to model order memory across scales. Participants performed JORs on lists of nouns (Experiment 1: list lengths = 4, 6, 8, 10) or consonants (Experiment 2: list lengths = 4, 8). In addition to the usual distance, primacy, and recency effects, instructions interacted with serial position of the later probe in both experiments, not only in response time, but also in error rate, suggesting that availability, not just accessibility, is affected by instructions. The congruity effect challenges current memory models. We fitted Hacker’s (Journal of Experimental Psychology: Human Learning and Memory, 6(6), 651–675, 1980) self-terminating search model to our data and found that a switch in search direction could explain the congruity effect for short lists, but not longer lists. This suggests that JORs may need to be understood via direct-access models, adapted to produce a congruity effect, or a mix of mechanisms.

Background Little is known about diet quality with a reduced-energy, low-fat, partial meal replacement plan, especially in individuals with type 2 diabetes. The Action for Health in Diabetes (Look AHEAD) trial implemented a partial meal replacement plan in the Intensive Lifestyle Intervention. Objective To compare dietary intake and percent meeting fat-related and food group dietary recommendations in Intensive Lifestyle Intervention and Diabetes Support and Education groups at 12 months. Design A randomized controlled trial comparing Intensive Lifestyle Intervention with Diabetes Support and Education at 0 and 12 months. Participants/setting From 16 US sites, the first 50% of participants (aged 45 to 76 years, overweight or obese, with type 2 diabetes) were invited to complete dietary assessments. Complete 0- and 12-month dietary assessments (collected between 2001 and 2004) were available for 2,397 participants (46.6% of total participants), with 1,186 randomized to Diabetes Support and Education group and 1,211 randomized to Intensive Lifestyle Intervention group. Main outcome measures A food frequency questionnaire assessed intake: energy; percent energy from protein, fat, carbohydrate, polyunsaturated fatty acids, and saturated fats; trans -fatty acids; cholesterol; fiber; weekly meal replacements; and daily servings from food groups from the Food Guide Pyramid. Statistical analyses performed Mixed-factor analyses of covariance, using Proc MIXED with a repeated statement, with age, sex, race/ethnicity, education, and income controlled. Unadjusted χ 2 tests compared percent meeting fat-related and food group recommendations at 12 months. Results At 12 months, Intensive Lifestyle Intervention participants had a significantly lower fat and cholesterol intake and greater fiber intake than Diabetes Support and Education participants. Intensive Lifestyle Intervention participants consumed more servings per day of fruits; vegetables; and milk, yogurt, and cheese; and fewer servings per day of fats, oils, and sweets than Diabetes Support and Education participants. A greater percentage of Intensive Lifestyle Intervention participants than Diabetes Support and Education participants met fat-related and most food group recommendations. Within Intensive Lifestyle Intervention, a greater percentage of participants consuming two or more meal replacements per day than participants consuming less than one meal replacement per day met most fat-related and food group recommendations. Conclusions The partial meal replacement plan consumed by Intensive Lifestyle Intervention participants was related to superior diet quality.

Background: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined. Methods: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model. Results: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling. Conclusions: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

Statins, routinely used to treat hypercholesterolemia, selectively induce apoptosis in some tumor cells by inhibiting the mevalonate pathway. Recent clinical studies suggest that a subset of breast tumors is particularly susceptible to lipophilic statins, such as fluvastatin. To quickly advance statins as effective anticancer agents for breast cancer treatment, it is critical to identify the molecular features defining this sensitive subset. We have therefore characterized fluvastatin sensitivity by MTT assay in a panel of 19 breast cell lines that reflect the molecular diversity of breast cancer, and have evaluated the association of sensitivity with several clinicopathological and molecular features. A wide range of fluvastatin sensitivity was observed across breast tumor cell lines, with fluvastatin triggering cell death in a subset of sensitive cell lines. Fluvastatin sensitivity was associated with an estrogen receptor alpha (ERa)-negative, basal-like tumor subtype, features that can be scored with routine and/or strong preclinical diagnostics. To ascertain additional candidate sensitivity-associated molecular features, we mined publicly available gene expression datasets, identifying genes encoding regulators of mevalonate production, nonsterol lipid homeostasis, and global cellular metabolism, including the oncogene MYC. Further exploration of this data allowed us to generate a 10-gene mRNA abundance signature predictive of fluvastatin sensitivity, which showed preliminary validation in an independent set of breast tumor cell lines. Here, we have therefore identified several candidate predictors of sensitivity to fluvastatin treatment in breast cancer, which warrant further preclinical and clinical evaluation. Fil: Goard, Carolyn A.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; Canadá Fil: Chan Seng Yue, Michelle . University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; Canadá Fil: Mullen, Peter J.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá Fil: Quiroga, Ariel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigaciones en Física e Ingeniería del Centro de la Provincia de Buenos Aires; Argentina. University of Alberta; Canadá Fil: Wasylishen, Amanda R.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; Canadá Fil: Clendening, James W.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; Canadá Fil: Sendorek, Dorota H. S.. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; Canadá Fil: Haider, Syed. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; Canadá Fil: Lehner, Richard. University of Alberta; Canadá Fil: Boutros, Paul C.. University Of Toronto; Canadá. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; Canadá Fil: Penn, Linda Z.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; Canadá

Despite its central role in human cancer, MYC deregulation is insufficient by itself to transform cells. Because inherent mechanisms of neoplastic control prevent precancerous lesions from becoming fully malignant, identifying transforming alleles of MYC that bypass such controls may provide fundamental insights into tumorigenesis. To date, the only activated allele of MYC known is T58A, the study of which led to identification of the tumor suppressor FBXW7 and its regulator USP28 as a novel therapeutic target. In this study, we screened a panel of MYC phosphorylation mutants for their ability to promote anchorage-independent colony growth of human MCF10A mammary epithelial cells, identifying S71A/S81A and T343A/S344A/S347A/S348A as more potent oncogenic mutants compared with wild-type (WT) MYC. The increased cell-transforming activity of these mutants was confirmed in SH-EP neuroblastoma cells and in three-dimensional MCF10A acini. Mechanistic investigations initiated by a genome-wide mRNA expression analysis of MCF10A acini identified 158 genes regulated by the mutant MYC alleles, compared with only 112 genes regulated by both WT and mutant alleles. Transcriptional gain-of-function was a common feature of the mutant alleles, with many additional genes uniquely dysregulated by individual mutant. Our work identifies novel sites of negative regulation in MYC and thus new sites for its therapeutic attack. Cancer Res; 73(21); 6504–15. ©2013 AACR.

Divergence in gene regulation can play a major role in evolution. Here, we used a phylogenetic framework to measure mRNA profiles in 15 yeast species from the phylum Ascomycota and reconstruct the evolution of their modular regulatory programs along a time course of growth on glucose over 300 million years. We found that modules have diverged proportionally to phylogenetic distance, with prominent changes in gene regulation accompanying changes in lifestyle and ploidy, especially in carbon metabolism. Paralogs have significantly contributed to regulatory divergence, typically within a very short window from their duplication. Paralogs from a whole genome duplication (WGD) event have a uniquely substantial contribution that extends over a longer span. Similar patterns occur when considering the evolution of the heat shock regulatory program measured in eight of the species, suggesting that these are general evolutionary principles. DOI: http://dx.doi.org/10.7554/eLife.00603.001, eLife digest The incredible diversity of living creatures belies the fact that their genes are quite similar. In the 1970s Mary-Claire King and Allan Wilson proposed that a process called gene regulation—which determines when, where and how genes are expressed as proteins—is responsible for this diversity. Four decades later, the central role of gene regulation in evolution has been confirmed in a wide range of species including bacteria, fungi, flies and mammals, although the details remain poorly understood. In recent years it has been suggested that the duplication of genes—and sometimes the duplication of whole genomes—has had a crucial influence on the part played by gene regulation in the evolution of many different species. Ascomycota fungi are uniquely suited to the study of genetics and evolution because of their diversity—they include C. albicans, a fungus that is found in the human mouth and gut, and various species of yeast—and because many of their genomes have already been sequenced. Moreover, their genomes are relatively small, which simplifies the task of working out how it has changed over the course of evolution. It is also known that species in this branch of the tree of life diverged before and after an event in which a whole genome was duplicated. Ascomycota fungi use glucose as a source of carbon in different ways during aerobic growth. Most, including C. albicans, are respiratory and rely on oxidative phosphorylation processes to produce energy. However, a small number—including S. cerevisiae and S. pombe, two types of yeast that are widely used as model organisms—prefer to ferment glucose, even when oxygen is available. Species that favor the latter respiro-fermentative lifestyle have evolved independently at least twice: once after the whole genome duplication event that lead to S. cerevisiae, and once when S. pombe and the other fission yeasts evolved. Thompson et al. have measured mRNA profiles in 15 different species of yeast and reconstructed how the regulation of groups of genes (modules) have evolved over a period of more than 300 million years. They found that modules have diverged proportionally to evolutionary time, with prominent changes in gene regulation being associated with changes in lifestyle (especially changes in carbon metabolism) and a whole genome duplication event. Gene duplication events result in gene paralogs—identical genes at different places in the genome—and these have made significant contributions to the evolution of different forms of gene regulation, especially just after the duplication event. Moreover, the paralogs produced in whole genome duplication events have resulted in bigger changes over longer periods of time. Similar patterns were observed in the regulation of the genes involved in the response to heat shock in eight of the species, which suggests that these are general evolutionary principles. The changes in gene expression associated with the respiro-fermentative lifestyle may also have implications for our understanding of cancer: healthy cells rely on oxidative phosphorylation to produce energy whereas, similar to yeast cells, most cancerous cells rely on respiro-fermentation. Furthermore, yeast cells and cancer cells both support their rapid growth and proliferation by using glucose for biosynthesis to support cell division, although this process is not fully understood. Normal cells, on the other hand, use glucose primarily for energy and tend not to divide rapidly. Thompson et al. found that the genes encoding enzymes in two biosynthetic pathways—one that produces the nucleotides necessary for DNA replication, and one that synthesizes glycine—are induced in respiro-fermentative yeasts but repressed in respiratory yeast cells. The fact that similar changes are observed in the same two pathways when normal cells become cancer cells suggests that these pathways have an important role in the development of cancer. The framework developed by Thompson et al. could also be used to explore the evolution of gene regulation in other species and biological processes. DOI: http://dx.doi.org/10.7554/eLife.00603.002

Divergence in gene regulation can play a major role in evolution. Here, we used a phylogenetic framework to measure mRNA profiles in 15 yeast species from the phylum Ascomycota and reconstruct the evolution of their modular regulatory programs along a time course of growth on glucose over 300 million years. We found that modules have diverged proportionally to phylogenetic distance, with prominent changes in gene regulation accompanying changes in lifestyle and ploidy, especially in carbon metabolism. Paralogs have significantly contributed to regulatory divergence, typically within a very short window from their duplication. Paralogs from a whole genome duplication (WGD) event have a uniquely substantial contribution that extends over a longer span. Similar patterns occur when considering the evolution of the heat shock regulatory program measured in eight of the species, suggesting that these are general evolutionary principles. DOI: http://dx.doi.org/10.7554/eLife.00603.001

Belinostat is a hydroxamate class HDAC inhibitor that has demonstrated activity in peripheral T-cell lymphoma and is undergoing clinical trials for non-hematologic malignancies. We studied the pharmacokinetics of belinostat in hepatocellular carcinoma patients to determine the main pathway of metabolism of belinostat. The pharmacokinetics of belinostat in liver cancer patients were characterized by rapid plasma clearance of belinostat with extensive metabolism with more than 4-fold greater relative systemic exposure of major metabolite, belinostat glucuronide than that of belinostat. There was significant interindividual variability of belinostat glucuronidation. The major pathway of metabolism involves UGT1A1-mediated glucuronidation and a good correlation has been identified between belinostat glucuronide formation and glucuronidation of known UGT1A1 substrates. In addition, liver microsomes harboring UGT1A1*28 alleles have lower glucuronidation activity for belinostat compared to those with wildtype UGT1A1. The main metabolic pathway of belinostat is through glucuronidation mediated primarily by UGT1A1, a highly polymorphic enzyme. The clinical significance of this finding remains to be determined. Trial Registration ClinicalTrials.gov NCT00321594

We describe the presentation, management, and clinical outcome of a massive acinic cell carcinoma of the parotid gland. The primary tumor and blood underwent exome sequencing which revealed deletions in CDKN2A as well as PPP1R13B, which induces p53. A damaging nonsynonymous mutation was noted in EP300, a histone acetylase which plays a role in cellular proliferation. This study provides the first insights into the genetic underpinnings of this cancer. Future large-scale efforts will be necessary to define the mutational landscape of salivary gland malignancies to identify therapeutic targets and biomarkers of treatment failure.

We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch's postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient's tumor, make a single cell suspension, mix it in known proportions with human fibroblasts (AG1522) and dispense a known number of cells of the mixture into each well of Ultra Low Attachment (ULA) 96-well plates to agglomerate into 1 HS/well, each containing an average of Applying Koch's postulates: (1) Does the patient's tumor contain cells with CSC properties? Yes: (a) The HSs grow to a size consistent with containing a CSC, (b) those that grew contain cells expressing ≥ 1 OKSM factor(s), and (c) such HSs contain differentiated progeny of the initial CSC. (2) Can we isolate and propagate these cells? In progress: testing HS serial growth, dissociation and reculture. (3) Can these cells induce the tumor in vivo? In progress: testing by xenografting individual CSC-containing HSs in NSG mice. (4) Do these cells contain and express specific gene products that give them CSC properties? Yes: Demonstrated for Oct4 and Nanog. (5) If we disrupt these genes, do the cells lose their CSC properties? In progress: (shRNA). (6) If we eliminate the CSCs do we eliminate the cancer? Yes: tested by (a) mathematical modeling of single CSC growth, differentiation of some progeny into ATCs capable of a limited number of divisions, that then become terminal non-proliferative tumor cells in the HSA; if a single CSC survives/remains, the HS will grow, i.e., the cancer will recur (63% for an average of 1 CSC in a HS, with a Poisson distribution, assuming that niche sites remain available), and (b) by measuring surviving fractions after exposure to radiation and/or chemotherapy agents (where sterilization of the CSC prevents the HS from growing ≥ 10 divisions. The HSA was applied to tumor samples taken from individual endometrial adenocarcinoma patients and correctly predicted, based on CSC radioresistance, which patients would fail their standard-of-care treatments. Conclusion: The CSC hypothesis is validated in the HSA. Citation Format: Christopher S. Lange, Talal Syed, Mike Zhang, Christian Sabalvaro, Hana I. Lim, Ghadir Salame, Bhargava Chitti, Ifeanyi Ilonzo, Arsalaan Ansari, Michelle Chan, Catherine Li, Jing Xu, Xiaotong Chen. Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3346.