Detection of BRCA1, BRCA2, and ATM Alterations in Matched Tumor Tissue and Circulating Tumor DNA in Patients with Prostate Cancer Screened in PROfound.

PURPOSE: Not all patients with metastatic castration-resistant prostate cancer (mCRPC) have sufficient tumor tissue available for multigene molecular testing. Furthermore, samples may fail because of difficulties within the testing procedure. Optimization of screening techniques may reduce failure rates; however, a need remains for additional testing methods to detect cancers with alterations in homologous recombination repair genes. We evaluated the utility of plasma-derived circulating tumor DNA (ctDNA) in identifying deleterious BRCA1, BRCA2 (BRCA), and ATM alterations in screened patients with mCRPC from the phase III PROfound study. PATIENTS AND METHODS: Tumor tissue samples were sequenced prospectively at Foundation Medicine, Inc. (FMI) using an investigational next-generation sequencing (NGS) assay based on FoundationOne®CDx to inform trial eligibility. Matched ctDNA samples were retrospectively sequenced at FMI, using an investigational assay based on FoundationOne®Liquid CDx. RESULTS: 81% (503/619) of ctDNA samples yielded an NGS result, of which 491 had a tumor tissue result. BRCA and ATM status in tissue compared with ctDNA showed 81% positive percentage agreement and 92% negative percentage agreement, using tissue as reference. At variant-subtype level, using tissue as reference, concordance was high for nonsense (93%), splice (87%), and frameshift (86%) alterations but lower for large rearrangements (63%) and homozygous deletions (27%), with low ctDNA fraction being a limiting factor. CONCLUSIONS: We demonstrate that ctDNA can greatly complement tissue testing in identifying patients with mCRPC and BRCA or ATM alterations who are potentially suitable for receiving targeted PARP inhibitor treatments, particularly patients with no or insufficient tissue for genomic analyses.

Disparities in Adverse Event Reporting for Hospitalized Children.

OBJECTIVES: Hospitals rely on voluntary event reporting (VER) for adverse event (AE) identification, although it captures fewer events than a trigger tool, such as Global Assessment of Pediatric Patient Safety (GAPPS). Medical providers exhibit bias based on patient weight status, race, and English proficiency. We compared the AE rate identified by VER with that identified using the GAPPS between hospitalized children by weight category, race, and English proficiency. METHODS: We identified a cohort of patients 2 years to younger than 18 years consecutively discharged from an academic children's hospital between June and October 2018. We collected data on patient weight status from age, sex, height, and weight, race/ethnicity by self-report, and limited English proficiency by record of interpreter use. We reviewed each chart with the GAPPS to identify AEs and reviewed VER entries for each encounter. We calculated an AE rate per 1000 patient-days using each method and compared these using analysis of variance. RESULTS: We reviewed 834 encounters in 680 subjects; 262 (38.5%) had overweight or obesity, 144 (21.2%) identified as Black, and 112 (16.5%) identified as Hispanic; 82 (9.8%) of encounters involved an interpreter. We identified 288 total AEs, 270 (93.8%) by the GAPPS and 18 (6.3%) by VER. A disparity in AE reporting was found for children with limited English proficiency, with fewer AEs by VER ( P = 0.03) compared with no difference in AEs by GAPPS. No disparities were found by weight category or race. CONCLUSIONS: Voluntary event reporting may systematically underreport AEs in hospitalized children with limited English proficiency.

Body Mass Index Category and Adverse Events in Hospitalized Children.

OBJECTIVE: To identify associations between patient body mass index (BMI) category and adverse event (AE) rate, severity, and preventability in a cohort of children discharged from an academic children's hospital. METHODS: We identified patients 2 to 17 years old consecutively discharged between June and October 2018. Patient age, sex, height, and weight were used to categorize patients as having underweight, normal weight, overweight, or obesity. We used the Global Assessment of Pediatrics Patient Safety trigger tool to identify AEs, which were scored for harm and preventability. The primary outcome was the rate of AEs; these were compared with Poisson regression. We used multivariable logistic regression to model event preventability. RESULTS: We reviewed 834 encounters in 680 subjects; 51 (7.5%) had underweight, 367 (54.0%) had normal weight, 112 (16.5%) had overweight, and 150 (22.1%) had obesity. Our cohort experienced 270 AEs, with an overall rate of 69.7 (61.8-78.5) AEs per 1000 patient-days: 67.7 (46.4-98.7) in underweight, 70.0 (59.4-82.4) in normal weight, 58.6 (42.5-79.7) in overweight, and 80.4 (62.5-103.6) in obesity, P = .46. No associations were seen between BMI category and AE severity. Children with obesity had an increased rate of preventable AEs (P < .01), but this association did not persist in the multivariable model. CONCLUSIONS: In this single-center study, we did not find associations between BMI category and rate, severity, or preventability of AEs.

The APT complex is involved in non-coding RNA transcription and is distinct from CPF.

The 3'-ends of eukaryotic pre-mRNAs are processed in the nucleus by a large multiprotein complex, the cleavage and polyadenylation factor (CPF). CPF cleaves RNA, adds a poly(A) tail and signals transcription termination. CPF harbors four enzymatic activities essential for these processes, but how these are coordinated remains poorly understood. Several subunits of CPF, including two protein phosphatases, are also found in the related 'associated with Pta1' (APT) complex, but the relationship between CPF and APT is unclear. Here, we show that the APT complex is physically distinct from CPF. The 21 kDa Syc1 protein is associated only with APT, and not with CPF, and is therefore the defining subunit of APT. Using ChIP-seq, PAR-CLIP and RNA-seq, we show that Syc1/APT has distinct, but possibly overlapping, functions from those of CPF. Syc1/APT plays a more important role in sn/snoRNA production whereas CPF processes the 3'-ends of protein-coding pre-mRNAs. These results define distinct protein machineries for synthesis of mature eukaryotic protein-coding and non-coding RNAs.

Architecture of eukaryotic mRNA 3'-end processing machinery.

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3' ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four ß propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3'-end processing.

RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7.

At the 3' ends of protein-coding genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine residues (Tyr1) of its C-terminal domain (CTD). In addition, the associated cleavage-and-polyadenylation factor (CPF) cleaves the transcript and adds a poly(a) tail. Whether these events are coordinated and how they lead to transcription termination remains poorly understood. Here we show that CPF from Saccharomyces cerevisiae is a Pol II-CTD phosphatase and that the CPF subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, the activity of Glc7 is required for normal Tyr1 dephosphorylation at the polyadenylation site, for recruitment of termination factors Pcf11 and Rtt103 and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing by CPF and transcription termination are coupled via Glc7-dependent Pol II-Tyr1 dephosphorylation.

Change in heat capacity for enzyme catalysis determines temperature dependence of enzyme catalyzed rates.

The increase in enzymatic rates with temperature up to an optimum temperature (Topt) is widely attributed to classical Arrhenius behavior, with the decrease in enzymatic rates above Topt ascribed to protein denaturation and/or aggregation. This account persists despite many investigators noting that denaturation is insufficient to explain the decline in enzymatic rates above Topt. Here we show that it is the change in heat capacity associated with enzyme catalysis (ΔC(‡)p) and its effect on the temperature dependence of ΔG(‡) that determines the temperature dependence of enzyme activity. Through mutagenesis, we demonstrate that the Topt of an enzyme is correlated with ΔC(‡)p and that changes to ΔC(‡)p are sufficient to change Topt without affecting the catalytic rate. Furthermore, using X-ray crystallography and molecular dynamics simulations we reveal the molecular details underpinning these changes in ΔC(‡)p. The influence of ΔC(‡)p on enzymatic rates has implications for the temperature dependence of biological rates from enzymes to ecosystems.

The crystal structure of the intact E. coli RelBE toxin-antitoxin complex provides the structural basis for conditional cooperativity.

The bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution, comprising both the RelB-inhibited RelE and the RelB dimerization domain that binds DNA. RelE and RelB associate into a V-shaped heterotetrameric complex with the ribbon-helix-helix (RHH) dimerization domain at the apex. Our structure supports a model in which relO is optimally bound by two adjacent RelB2E heterotrimeric units, and is not compatible with concomitant binding of two RelB2E2 heterotetramers. The results thus provide a firm basis for understanding the model of conditional cooperativity at the molecular level.