Reconstructing and reprogramming the tumor-propagating potential of glioblastoma stem-like cells.

Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to "induced" TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies. PAPERCLIP:

A negative feedback loop of transcription factors specifies alternative dendritic cell chromatin States.

During hematopoiesis, cells originating from the same stem cell reservoir differentiate into distinct cell types. The mechanisms enabling common progenitors to differentiate into alternative cell fates are not fully understood. Here, we identify cell-fate-determining transcription factors (TFs) governing dendritic cell (DC) development by annotating the enhancer landscapes of the DC lineage. Combining these analyses with detailed overexpression, knockdown, and ChIP-Seq studies, we show that Irf8 functions as a plasmacytoid DC epigenetic and fate-determining TF, regulating massive, cell-specific chromatin changes in thousands of pDC enhancers. Importantly, Irf8 forms a negative feedback loop with Cebpb, a monocyte-derived DC epigenetic fate-determining TF. We show that using this circuit logic, a pulse of TF expression can stably define epigenetic and transcriptional states, regardless of the microenvironment. More broadly, our study proposes a general paradigm that allows closely related cells with a similar set of signal-dependent factors to generate differential and persistent enhancer landscapes.

The RNA structurome in the asexual blood stages of malaria pathogen plasmodium falciparum.

Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite's asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5' UTR, CDS and 3' UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs. Abbreviations: CDS: Coding Sequence; DNA: Deoxyribonucleic Acid; dsRNA: double-stranded RNA; IDC: Intra-erythrocytic Developmental Cycle (IDC); m6A: N6-methyladenosine; mRNA: Messenger RNA; ncRNA: Non-coding RNA; RBC: Red Blood cells; RBP: RNA-Binding Protein; REC: Relative Expression Counts; RNA-seq: RNA-sequencing; RNA: Ribonucleic Acid; RNP: Ribonucleoprotein; RPKM: Reads Per Kilobase of transcript Per Million; rRNA: Ribosomal RNA 16. RUFs: RNAs of Unknown Function; SHAPE: Selective 2'-hydroxyl acylation analysed by primer extension; snoRNA: Small Nucleolar RNA; snRNA: Small Nuclear RNA; SRP-RNA: Signal Recognition Particle RNA; ssRNA: (Single-stranded RNA); TE: Translation Efficiency; tRNA: transfer RNA; UTR: Untranslated Region.

Quantitative next-generation sequencing-based analysis indicates progressive accumulation of microsatellite instability between atypical hyperplasia/endometrial intraepithelial neoplasia and paired endometrioid endometrial carcinoma.

Atypical hyperplasia/endometrial intraepithelial neoplasia is an accepted precursor to endometrioid-type endometrial carcinoma. Mismatch repair-deficient endometrial carcinomas are also known to be a biologically and clinically distinct subset of tumors. However, the development of microsatellite instability in endometrial carcinogenesis has not yet been evaluated by novel next-generation sequencing-based methods. We examined 17 mismatch repair-deficient endometrioid endometrial carcinomas and their paired atypical hyperplasia/endometrial intraepithelial neoplasia precursors using a next-generation sequencing panel with quantitative microsatellite instability detection at 336 loci. Findings were compared to histological features, polymerase chain reaction-based microsatellite instability testing, immunohistochemical expression of mismatch repair proteins, and tumor mutational burden calculations. All 17 endometrial carcinomas and 8/17 atypical hyperplasia/endometrial intraepithelial neoplasia showed microsatellite instability by next-generation sequencing-based testing. Endometrial carcinoma specimens showed significantly more unstable microsatellite loci than paired atypical hyperplasia/endometrial intraepithelial neoplasia (mean: 40.0% vs 19.9 unstable loci, respectively). Out of nine microsatellite-stable atypical hyperplasia/endometrial intraepithelial neoplasia specimens, four showed mismatch repair loss by immunohistochemistry. All atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens with microsatellite instability were also mismatch repair-deficient by immunohistochemistry. Tumor mutational burden was significantly greater in endometrial carcinoma than in paired atypical hyperplasia/endometrial intraepithelial neoplasia specimens, and tumor mutational burden was significantly correlated with percent unstable microsatellite loci. Paired atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens show progressive accumulation of unstable microsatellite loci following loss of mismatch repair protein expression. Comprehensive next-generation sequencing-based testing of endometrial carcinomas offers new insights into endometrial carcinogenesis and opportunities for improved tumor surveillance, diagnosis, and management.

Defining the 5΄ and 3΄ landscape of the Drosophila transcriptome with Exo-seq and RNaseH-seq.

Cells regulate biological responses in part through changes in transcription start sites (TSS) or cleavage and polyadenylation sites (PAS). To fully understand gene regulatory networks, it is therefore critical to accurately annotate cell type-specific TSS and PAS. Here we present a simple and straightforward approach for genome-wide annotation of 5΄- and 3΄-RNA ends. Our approach reliably discerns bona fide PAS from false PAS that arise due to internal poly(A) tracts, a common problem with current PAS annotation methods. We applied our methodology to study the impact of temperature on the Drosophila melanogaster head transcriptome. We found hundreds of previously unidentified TSS and PAS which revealed two interesting phenomena: first, genes with multiple PASs tend to harbor a motif near the most proximal PAS, which likely represents a new cleavage and polyadenylation signal. Second, motif analysis of promoters of genes affected by temperature suggested that boundary element association factor of 32 kDa (BEAF-32) and DREF mediates a transcriptional program at warm temperatures, a result we validated in a fly line where beaf-32 is downregulated. These results demonstrate the utility of a high-throughput platform for complete experimental and computational analysis of mRNA-ends to improve gene annotation.

High prevalence of MiTF staining in undifferentiated pleomorphic sarcoma: caution in the use of melanocytic markers in sarcoma.

AIMS: The diagnosis of undifferentiated pleomorphic sarcoma (UPS) may be challenging, as other lesions with undifferentiated spindle cell morphology must be excluded, including melanoma. Microphthalmia-associated transcription factor (MiTF) stains naevi and epithelioid melanomas, as well as some mesenchymal neoplasms. The aim of this study was to evaluate the prevalence of MiTF and melanocytic markers in UPS and a subset of atypical fibroxanthoma (AFX). METHODS AND RESULTS: MiTF, SOX10, Melan-A, HMB45 and S100 immunostaining was performed on resection specimens from 19 UPSs and five AFXs. Next-generation sequencing of 50 genes was performed in UPSs to exclude dedifferentiated melanoma. In 17 of 19 UPSs (89%), tumour cells showed nuclear positivity for MiTF that was not eliminated by casein block. Three showed focal nuclear staining for HMB45, which was eliminated by casein block. One showed focal nuclear vacuole staining for S100 with red but not brown chromogen. None expressed SOX10 or Melan-A. Mutational analysis of 15 UPSs with adequate DNA showed no mutations within hotspot regions of BRAF, KIT, or NRAS. Four of five AFXs (80%) stained with MiTF; other markers were negative. CONCLUSION: There is a high prevalence of nuclear MiTF expression in UPSs (89%) and AFXs (80%). Rare UPSs showed non-specific nuclear HMB45 or S100 staining. These findings argue against using MiTF in isolation to differentiate between UPS or AFX and melanoma, and caution in interpreting focal staining for a single additional melanocytic marker. Casein block may eliminate non-specific staining. MiTF should be used to support a diagnosis of melanoma only if multiple melanocytic markers are positive.

Polyglucosan bodies in intramuscular nerve branches are a poor predictor of GBE1 mutation and adult polyglucosan body disease.

INTRODUCTION: Adult polyglucosan body disease (APBD) is associated with formation of polyglucosan bodies in peripheral nerve branches. Some muscle biopsies show these inclusions in intramuscular nerve branches. It has not been established whether the presence of multiple polyglucosan bodies in intramuscular peripheral nerve branches could or should suggest testing for APBD. METHODS: Fifteen muscle biopsies from adults between the ages of 36 and 84 years, all showing polyglucosan bodies in intramuscular peripheral nerve twigs, were tested by sequencing of the GBE1 gene. RESULTS: In 4 patients, testing identified heterozygous missense mutations not previously described. No homozygous or compound heterozygous mutations were identified. CONCLUSIONS: The presence of polyglucosan bodies in intramuscular nerve twigs by itself, even if they are multiple, is not an indication of APBD. Further testing may only be indicated in patients with clinical disease manifestations.

Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays.

Genomewide loss-of-function (LOF) screening using clustered regularly interspaced short palindromic repeats (CRISPR) has facilitated the discovery of novel gene functions across diverse physiological and pathophysiological systems. A challenge with conventional genomewide CRISPR-Cas9 libraries is the unwieldy size (60,000-120,000 constructs), which is resource intensive and prohibitive in some experimental contexts. One solution to streamlining CRISPR screening is by multiplexing two or more guides per gene on a single construct, which enables functional redundancy to compensate for suboptimal gene knockout by individual guides. In this regard, AsCas12a (Cpf1) and its derivatives, for example, enhanced AsCas12a (enAsCas12a), have enabled multiplexed guide arrays to be specifically and efficiently processed for genome editing. Prior studies have established that multiplexed CRISPR-Cas12a libraries perform comparably to the larger equivalent CRISPR-Cas9 libraries, yet the most efficient CRISPR-Cas12a library design remains unresolved. In this study, we demonstrate that CRISPR-Cas12a genomewide LOF screening performed optimally with three guides arrayed per gene construct and could be adapted to robotic cell culture without noticeable differences in screen performance. Thus, the conclusions from this study provide novel insight to streamlining genomewide LOF screening using CRISPR-Cas12a and robotic cell culture.

Recommendations for the Use of in Silico Approaches for Next-Generation Sequencing Bioinformatic Pipeline Validation: A Joint Report of the Association for Molecular Pathology, Association for Pathology Informatics, and College of American Pathologists.

In silico approaches for next-generation sequencing (NGS) data modeling have utility in the clinical laboratory as a tool for clinical assay validation. In silico NGS data can take a variety of forms, including pure simulated data or manipulated data files in which variants are inserted into existing data files. In silico data enable simulation of a range of variants that may be difficult to obtain from a single physical sample. Such data allow laboratories to more accurately test the performance of clinical bioinformatics pipelines without sequencing additional cases. For example, clinical laboratories may use in silico data to simulate low variant allele fraction variants to test the analytical sensitivity of variant calling software or simulate a range of insertion/deletion sizes to determine the performance of insertion/deletion calling software. In this article, the Working Group reviews the different types of in silico data with their strengths and limitations, methods to generate in silico data, and how data can be used in the clinical molecular diagnostic laboratory. Survey data indicate how in silico NGS data are currently being used. Finally, potential applications for which in silico data may become useful in the future are presented.

Containers in Bioinformatics: Applications, Practical Considerations, and Best Practices in Molecular Pathology.

Systematic implementation of bioinformatics resources for next generation sequencing (NGS)-based clinical testing is an arduous undertaking. One of the key challenges involves developing an ecosystem of information technology infrastructure for enabling scalable and reproducible bioinformatics services that is resilient and secure for handling genetic and protected health information, often embedded in an existing non-bioinformatics-oriented infrastructure. Container technology provides an ideal and infrastructure-agnostic solution for molecular laboratories developing and using bioinformatics pipelines, whether on-premise or using the cloud. A container is a technology that provides a consistent computational environment and enables reproducibility, scalability, and security when developing NGS bioinformatics analysis pipelines. Containers can increase the bioinformatics team's productivity by automating and simplifying the maintenance of complex bioinformatics resources, as well as facilitate validation, version control, and documentation necessary for clinical laboratory regulatory compliance. Although there is increasing popularity in adopting containers for developing NGS bioinformatics pipelines, there is wide variability and inconsistency in the usage of containers that may result in suboptimal performance and potentially compromise the security and privacy of protected health information. In this article, the authors highlight the current state and provide best or recommended practices for building, using containers in NGS bioinformatics solutions in a clinical setting with focus on scalability, optimization, maintainability, and data security.

A performance evaluation study: Variant annotation tools - the enigma of clinical next generation sequencing (NGS) based genetic testing.

Dramatically expanding our ability for clinical genetic testing for inherited conditions and complex diseases such as cancer, next generation sequencing (NGS) technologies are allowing for rapid interrogation of thousands of genes and identification of millions of variants. Variant annotation, the process of assigning functional information to DNA variants based on the standardized Human Genome Variation Society (HGVS) nomenclature, is a fundamental challenge in the analysis of NGS data that has led to the development of many bioinformatic algorithms. In this study, we evaluated the performance of 3 variant annotation tools: Alamut® Batch, Ensembl Variant Effect Predictor (VEP), and ANNOVAR, benchmarked by a manually curated ground-truth set of 298 variants from the medical exome database at the Molecular Diagnostics Laboratory at Lurie Children's Hospital. Of the 3 tools, VEP produces the most accurate variant annotations (HGVS nomenclature for 297 of the 298 variants) due to usage of updated gene transcript versions within the algorithm. Alamut® Batch called 296 of the 298 variants correctly; strikingly, ANNOVAR exhibited the greatest number of discrepancies (20 of the 298 variants, 93.3% concordance with ground-truth set). Adoption of validated methods of variant annotation is critical in post-analytical phases of clinical testing.

Unified classification of mouse retinal ganglion cells using function, morphology, and gene expression.

Classification and characterization of neuronal types are critical for understanding their function and dysfunction. Neuronal classification schemes typically rely on measurements of electrophysiological, morphological, and molecular features, but aligning such datasets has been challenging. Here, we present a unified classification of mouse retinal ganglion cells (RGCs), the sole retinal output neurons. We use visually evoked responses to classify 1,859 mouse RGCs into 42 types. We also obtain morphological or transcriptomic data from subsets and use these measurements to align the functional classification to publicly available morphological and transcriptomic datasets. We create an online database that allows users to browse or download the data and to classify RGCs from their light responses using a machine learning algorithm. This work provides a resource for studies of RGCs, their upstream circuits in the retina, and their projections in the brain, and establishes a framework for future efforts in neuronal classification and open data distribution.

Transcript analysis for variant classification resolution in a child with primary ciliary dyskinesia.

Transcriptional analysis can be utilized to reconcile variants of uncertain significance, particularly those predicted to impact splicing. Laboratory analysis of the predicted mRNA transcript may allow inference of the in vivo impact of the variant and aid prediction of its clinical significance. We present a patient with classical features of primary ciliary dyskinesia (PCD) who was identified to have compound heterozygous variants in the DNAH11 gene (c.10691 + 2T > C, c.13523_13543dup21) via trio whole-exome sequencing in 2013. These variants were originally classified as Mutation and Likely Mutation. However, these variants were downgraded to variants of uncertain significance (VUSs) during reanalysis in 2016 because of uncertainty that they caused a loss of function of the gene. c.10691 + 2T > C is predicted to abrogate the canonical splice site and lead to the skipping of exon 65, but the adjoining of exon 64 and exon 66 in the DNAH11 transcript preserves the reading frame of the resultant protein. c.13523_13543dup21 is located in the last exon of the DNAH11 coding sequence, upstream of the canonical stop codon, which suggests a reduced likelihood to trigger nonsense-mediated decay (NMD). Transcriptional analysis was performed to characterize the impact of the variants, resulting in reclassification of c.10691 + 2T > C to Likely Pathogenic by providing evidence that it results in a deleterious effect and subsequent downstream reclassification of c.13523_13543dup21 to Likely Pathogenic as well. Our case illustrates the potential impact of transcriptional analysis on variant resolution, supporting its usage on variants that exert an unpredictable effect on splicing.

Genomic Autopsy of Sudden Deaths in Young Individuals.

Importance: Postmortem genetic testing of young individuals with sudden death has previously identified pathogenic gene variants. However, prior studies primarily considered highly penetrant monogenic variants, often without detailed decedent and family clinical information. Objective: To assess genotype and phenotype risk in a diverse cohort of young decedents with sudden death and their families. Design, Setting, and Participants: Pathological and whole-genome sequence analysis was conducted in a cohort referred from a national network of medical examiners. Cases were accrued prospectively from May 2015 to March 2019 across 24 US states. Analysis began September 2016 and ended November 2020. Exposures: Evaluation of autopsy and clinical data integrated with whole-genome sequence data and family member evaluation. Results: A total of 103 decedents (mean [SD] age at death, 23.7 [11.9] years; age range, 1-44 years), their surviving family members, and 140 sex- and genetic ancestry-matched controls were analyzed. Among 103 decedents, autopsy and clinical data review categorized 36 decedents with postmortem diagnoses, 23 decedents with findings of uncertain significance, and 44 with sudden unexplained death. Pathogenic/likely pathogenic (P/LP) genetic variants in arrhythmia or cardiomyopathy genes were identified in 13 decedents (12.6%). A multivariable analysis including decedent phenotype, ancestry, and sex demonstrated that younger decedents had a higher burden of P/LP variants and select variants of uncertain significance (effect size, -1.64; P = .001). These select, curated variants of uncertain significance in cardiac genes were more common in decedents than controls (83 of 103 decedents [86%] vs 100 of 140 controls [71%]; P = .005), and decedents harbored more rare cardiac variants than controls (2.3 variants per individual vs 1.8 in controls; P = .006). Genetic testing of 31 parent-decedent trios and 14 parent-decedent dyads revealed 8 transmitted P/LP variants and 1 de novo P/LP variant. Incomplete penetrance was present in 6 of 8 parents who transmitted a P/LP variant. Conclusions and Relevance: Whole-genome sequencing effectively identified P/LP variants in cases of sudden death in young individuals, implicating both arrhythmia and cardiomyopathy genes. Genomic analyses and familial phenotype association suggest potentially additive, oligogenic risk mechanisms for sudden death in this cohort.

Follicular Thyroid Neoplasms: Comparison of Clinicopathologic and Molecular Features of Atypical Adenomas and Follicular Thyroid Carcinomas.

In follicular thyroid neoplasms without invasion, a diagnosis of atypical adenoma (AA) (follicular tumor of uncertain malignant potential) may be rendered if atypical features (indefinite capsular/vascular invasion, necrosis, solid growth, increased mitoses) are present. This study compares clinical, histologic, and molecular features of patients with AAs (n=31), nonmetastatic follicular thyroid carcinoma (nmFTC) (n=18), and metastatic follicular thyroid carcinoma (mFTC) (n=38). Patients with mFTC were older. Mitotic activity in areas of solid growth was greatest in mFTC (P=0.05). Oncocytic tumors tended to show solid growth (P=0.04). The presence or frequency of capsular and/or vascular invasion was not different between nmFTC and mFTC. TERT promoter mutations were higher in patients with mFTC (50%) than nmFTC (25%) and AA (10%) (P=0.02). TERT promoter mutation was associated with necrosis (P=0.01) and solid growth plus increased mitoses (P=0.03). Necrosis and TERT promoter mutations were identified in all groups, most frequently in mFTC. The combination of solid growth with increased mitoses, necrosis, and TERT promoter mutation was only seen in follicular carcinomas. Poorly differentiated features, vascular invasion, and TERT promoter mutation correlated with metastasis in FTC. Given the low frequency of necrosis and TERT promoter mutation in AAs, close clinical follow-up is recommended in patients with these findings, especially if additional atypical features (such as solid growth plus mitoses) are present.

Next-generation sequencing identifies 2 genomically distinct groups among pyloric gland adenomas.

The molecular alterations identified among pyloric gland adenomas (PGAs) in the published literature are based on polymerase chain reaction of targeted genes, and next-generation sequencing (NGS) has not been performed. In this study, we performed NGS and correlated the molecular alterations with the histologic grade of dysplasia and immunohistochemical findings in a cohort of PGAs. Successful DNA extraction and sequencing were performed in 15 pyloric gland adenomas/adenocarcinoma from 12 patients. Additionally, 4 specimens of autoimmune gastritis were selected to serve as the control group. Ten PGAs with low-grade dysplasia were seen to have mutations in the triad of APC, KRAS, and GNAS genes. Five PGAs with high-grade dysplasia/adenocarcinoma exhibited mutations in several genes including APC, CTNNB1, KRAS, GNAS, TP53, CDKN2A, PIK3CA, and EPHA5 genes but did not exhibit mutations in the triad of APC, KRAS, and GNAS genes. The median tumor mutational burden was higher in PGAs with high-grade dysplasia/adenocarcinoma when compared with PGAs with low-grade dysplasia (5.25 and 4.38, respectively). PGAs with high-grade dysplasia/adenocarcinoma had more chromosomal gains and losses than PGAs with low-grade dysplasia. The molecular findings suggest that there are 2 separate mutator pathways of dysplasia development in PGAs.

Detailed Phenotypic and Functional Characterization of a Rare, Antibody-Dependent SLAM-Associated Protein Expression Pattern.

SLAM-associated protein (SAP) is an adaptor molecule that facilitates critical effector functions in immune cells, and its deficiency causes X-linked lymphoproliferative disease type 1 in which effector responses directed against EBV are severely compromised. The primary objective of this study was to phenotypically and functionally characterize a rare, CD8 T cell-restricted bimodal SAP expression pattern observed in healthy, human donors with the widely used 1C9-SAP mAb clone. We initially observed this pattern during the clinical validation of our flow cytometry-based assay to diagnose X-linked lymphoproliferative disease type 1 in our laboratory. For this validation study, we used multiparameter flow cytometry to identify cytosolic SAP expression in lymphocyte subsets, and CD8 T cells from the donors displaying the rare SAP expression pattern mentioned above were separately further evaluated by intracellular cytokine and CD107a staining to examine polyfunctionality following PMA/ionomycin and HLA class I allele-restricted EBV peptide epitope-induced T cell activation. Our data revealed that SAP 1C9-hi CD8 T cells clearly displayed higher polyfunctional responses versus SAP 1C9-lo CD8 T cells following PMA/ionomycin stimulation. Furthermore, polyfunctional EBV-specific CD8 T cell responses segregated with the SAP 1C9-hi CD8 T cells and not the SAP 1C9-lo CD8 T cells. Additionally, and rather intriguingly, short- and long-term T cell stimulation selectively diminished the signal for the 1C9-hi subset. Overall, our data suggest that although rare, this unique SAP expression pattern merits further evaluation as it has the potential to provide some insight into fundamental processes as they might relate to host-pathogen dynamics.

A phase 1 study of azacitidine with high-dose cytarabine and mitoxantrone in high-risk acute myeloid leukemia.

In this phase 1 study, azacitidine (AZA) was given before high-dose cytarabine (HiDAC) and mitoxantrone (mito) based on the hypothesis that epigenetic priming with a hypomethylating agent before cytotoxic chemotherapy would improve response rates in patients with high-risk acute myeloid leukemia (AML), including relapsed/refractory disease. The primary objective was to establish the recommended phase 2 dose of AZA given before standard HiDAC/mito. In a dose escalation scheme, 46 patients (median age, 66 years) received AZA at 37.5, 50, or 75 mg/m2 subcutaneously or IV once daily on days 1 to 5 followed by HiDAC (3000 mg/m2) and mitoxantrone (30 mg/m2) once each on days 6 and 10 (the HiDAC/mito dose was reduced 33% in elderly subjects). Two dose-limiting toxicities occurred (both in the same patient): acute liver failure and kidney injury at the 50 mg/m2 dose. The 30-day induction death rate was 2.2% (1 of 46). The overall response rate, including complete remission and complete remission with incomplete count recovery, was 61% (28 of 46). Previously untreated patients aged ≥60 years with therapy-related AML and de novo AML were more likely to respond than untreated patients with AML progressing from an antecedent hematologic disorder (myelodysplastic syndrome and chronic myelomonocytic leukemia). Patients with favorable European Leukemia Network risk (P = .008), NPM1 mutations (P = .007), or IDH2 mutations (P = .03) were more likely to respond, and those with TP53 mutations (P = .03) were less likely to respond. The recommended phase 2 dose of AZA is 75 mg/m2 per day on days 1 to 5 followed by HiDAC (3000 mg/m2) and mitoxantrone (30 mg/m2) once each on days 6 and 10. This trial was registered at www.clinicaltrials.gov as #NCT01839240.

HHMMiR: efficient de novo prediction of microRNAs using hierarchical hidden Markov models.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding single-stranded RNAs (20-23 nts) that are known to act as post-transcriptional and translational regulators of gene expression. Although, they were initially overlooked, their role in many important biological processes, such as development, cell differentiation, and cancer has been established in recent times. In spite of their biological significance, the identification of miRNA genes in newly sequenced organisms is still based, to a large degree, on extensive use of evolutionary conservation, which is not always available. RESULTS: We have developed HHMMiR, a novel approach for de novo miRNA hairpin prediction in the absence of evolutionary conservation. Our method implements a Hierarchical Hidden Markov Model (HHMM) that utilizes region-based structural as well as sequence information of miRNA precursors. We first established a template for the structure of a typical miRNA hairpin by summarizing data from publicly available databases. We then used this template to develop the HHMM topology. CONCLUSION: Our algorithm achieved average sensitivity of 84% and specificity of 88%, on 10-fold cross-validation of human miRNA precursor data. We also show that this model, trained on human sequences, works well on hairpins from other vertebrate as well as invertebrate species. Furthermore, the human trained model was able to correctly classify ~97% of plant miRNA precursors. The success of this approach in such a diverse set of species indicates that sequence conservation is not necessary for miRNA prediction. This may lead to efficient prediction of miRNA genes in virtually any organism.

insiM: in silico Mutator Software for Bioinformatics Pipeline Validation of Clinical Next-Generation Sequencing Assays.

Lack of reliable reference samples containing different mutations of interest across large sets of disease-relevant loci limits the extensive validation clinical next-generation sequencing (NGS) assays and their associated bioinformatics pipelines. Herein, we have generated a publicly available, highly flexible tool, in silico Mutator (insiM), to introduce point mutations, insertions, deletions, and duplications of any size into real data sets of amplicon-based or hybrid-capture NGS assays. insiM accepts an alignment file along with target territory and produces paired-end FASTQ files containing specified mutations via modification of original sequencing reads. Mutant signal is, thus, generated within the context of existing real-world data to most closely mimic assay performance. Resulting files may then be passed through the assay's bioinformatics pipeline to assist with assay/bioinformatics validation and to identify performance gaps in detection. To establish the basic functionality of the software, a series of simulation experiments with varying mutation types, sizes, and allele frequencies were performed across the entire clinical territory of hybrid-capture and amplicon-based clinical assays developed at The University of Chicago. This work demonstrates the utility of insiM as a supplementary tool during the validation of an NGS assay's bioinformatics pipeline.