Low frequency of allergy referral for penicillin allergy evaluation in an urban Boston primary care setting.

Background: A key strategy to combat the public health crisis of antimicrobial resistance is to use appropriate antibiotics, which is difficult in patients with a penicillin allergy label. Objective: Our aim was to investigate racial and ethnic differences related to penicillin allergy labeling and referral to allergy/immunology in primary care. Methods: This was a retrospective study of Tufts Medical Center's Boston-based primary care patients in 2019. Univariable and multivariable logistic regression models were used to examine demographic associations with (1) penicillin allergy label and (2) allergist referral. Results: Of 21,918 primary care patients, 2,391 (11%) had a penicillin allergy label; of these, 249 (10%) had an allergist referral. In multivariable logistic regression models, older age (adjusted odds ratio [aOR] = 1.06 [95% CI = 1.04-1.09]) and female sex (aOR = 1.58 [95% CI = 1.44-1.74]) were associated with higher odds of penicillin allergy label carriage. Black race (aOR = 0.77 [95% CI = 0.69-0.87]) and Asian race (aOR = 0.47 [95% CI = 0.41-0.53]) were associated with lower odds of penicillin allergy label carriage. In multivariable regression, allergist referral was associated with female sex (aOR = 1.52 [95% CI = 1.10-2.10]) and Black race (aOR = 1.74 [95% CI = 1.25-2.45]). Of 93 patients (37%) who completed their allergy visit, 26 (28%) had received penicillin allergy evaluation or were scheduled to receive a penicillin allergy evaluation at a future visit. Conclusions: There were racial differences in penicillin allergy labeling and referral. Allergy referral for penicillin allergy assessment was rare. Larger studies are needed to assess penicillin allergy labeling and delabeling with an equity focus on optimizing patient health outcomes.

Characterization of Phosphopantetheinyl Hydrolase from Mycobacterium tuberculosis.

Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4'-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis's phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium's cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.

Macrocyclic Peptides that Selectively Inhibit the Mycobacterium tuberculosis Proteasome.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

The DnaK Chaperone System Buffers the Fitness Cost of Antibiotic Resistance Mutations in Mycobacteria.

Chaperones aid in protein folding and maintenance of protein integrity. In doing so, they have the unique ability to directly stabilize resistance-conferring amino acid substitutions in drug targets and to counter the stress imparted by these substitutions, thus supporting heritable antimicrobial resistance (AMR). We asked whether chaperones support AMR in Mycobacterium smegmatis, a saprophytic model of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). We show that DnaK associates with many drug targets and that DnaK associates more with AMR-conferring mutant RNA polymerase (RNAP) than with wild-type RNAP. In addition, frequency-of-resistance (FOR) and fitness studies reveal that the DnaK system of chaperones supports AMR in antimicrobial targets in mycobacteria, including RNAP and the ribosome. These findings highlight chaperones as potential targets for drugs to overcome AMR in mycobacteria, including M. tuberculosis, as well as in other pathogens.IMPORTANCE AMR is a global problem, especially for TB. Here, we show that mycobacterial chaperones support AMR in M. smegmatis, a nonpathogenic model of M. tuberculosis, the causative agent of TB. In particular, the mycobacterial DnaK system of chaperones supports AMR in the antimicrobial targets RNA polymerase and the ribosome. This is the first report showing a role for protein chaperones in mediating AMR in mycobacteria. Given the widespread role of protein chaperones in enabling genomic diversity, we anticipate that our findings can be extended to other microbes.

Isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of MYBL1 or MYB and a benign disease course.

The "isomorphic subtype of diffuse astrocytoma" was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.

A multicomponent intervention to improve adherence to opioid prescribing and monitoring guidelines in primary care.

OBJECTIVE: Guidelines for appropriate management of chronic opioid therapy are underutilized by primary care physicians (PCPs). The authors hypothesized that developing a multicomponent, team-based opioid management system with electronic health record (EHR) support would allow our clinicians to improve adherence to chronic opioid prescribing and monitoring guidelines. DESIGN: This was a retrospective pre-post study. SETTING: The authors performed this intervention at our large, urban, academic primary care practice. PATIENTS, PARTICIPANTS: All patients with the diagnosis of "chronic pain, opioid requiring (ICD-10 F11.20)" on their primary care EHR problem lists were included in this study. INTERVENTION: The authors implemented a five-pronged strategy to improve our system of opioid prescribing, including (1) a patient registry with regular dissemination of reports to PCPs; (2) standardization of policies regarding opioid prescribing and monitoring; (3) development of a risk-assessment algorithm and riskstratified monitoring guidelines; (4) a team-based approach to care with physician assistant care managers; and (5) an EHR innovation to facilitate communication and guideline adherence. MAIN OUTCOME MEASURES: The authors measured percent adherence to opioid prescribing guidelines, including annual patient-provider agreements, biannual urine drug screens (UDSs), and prescription monitoring program (PMP) verification. RESULTS: Between September 2015 and September 2016, the percentage of patients on chronic opioid therapy with a signed controlled substances agreement within the preceding year increased from 46 to 76 percent (p < 0.0001), while the percentage of patients with a UDS done within the past 6 months rose from 23 to 79 percent (p < 0.0001). The percentage of patients whose state PMPs profile had been checked by a primary care team member in the past year rose from 45 to 97 percent (p < 0.0001). CONCLUSION: A comprehensive strategy to standardize chronic opioid prescribing in our primary care practice coincided with an increase in adherence to opioid management guidelines.

Tumors diagnosed as cerebellar glioblastoma comprise distinct molecular entities.

In this multi-institutional study we compiled a retrospective cohort of 86 posterior fossa tumors having received the diagnosis of cerebellar glioblastoma (cGBM). All tumors were reviewed histologically and subjected to array-based methylation analysis followed by algorithm-based classification into distinct methylation classes (MCs). The single MC containing the largest proportion of 25 tumors diagnosed as cGBM was MC anaplastic astrocytoma with piloid features representing a recently-described molecular tumor entity not yet included in the WHO Classification of Tumours of the Central Nervous System (WHO classification). Twenty-nine tumors molecularly corresponded to either of 6 methylation subclasses subsumed in the MC family GBM IDH wildtype. Further we identified 6 tumors belonging to the MC diffuse midline glioma H3 K27 M mutant and 6 tumors allotted to the MC IDH mutant glioma subclass astrocytoma. Two tumors were classified as MC pilocytic astrocytoma of the posterior fossa, one as MC CNS high grade neuroepithelial tumor with BCOR alteration and one as MC control tissue, inflammatory tumor microenvironment. The methylation profiles of 16 tumors could not clearly be assigned to one distinct MC. In comparison to supratentorial localization, the MC GBM IDH wildtype subclass midline was overrepresented, whereas the MCs GBM IDH wildtype subclass mesenchymal and subclass RTK II were underrepresented in the cerebellum. Based on the integration of molecular and histological findings all tumors received an integrated diagnosis in line with the WHO classification 2016. In conclusion, cGBM does not represent a molecularly uniform tumor entity, but rather comprises different brain tumor entities with diverse prognosis and therapeutic options. Distinction of these molecular tumor classes requires molecular analysis. More than 30% of tumors diagnosed as cGBM belong to the recently described molecular entity of anaplastic astrocytoma with piloid features.

Selective Phenylimidazole-Based Inhibitors of the Mycobacterium tuberculosis Proteasome.

Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Coevolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders nonreplicating Mtb susceptible to reactive nitrogen species in vitro and unable to survive in the lungs of mice, validating the Mtb proteasome as a promising target for anti-Mtb agents. Using a structure-guided and flow chemistry-enabled study of structure-activity relationships, we developed phenylimidazole-based peptidomimetics that are highly potent for Mtb20S. X-ray structures of selected compounds with Mtb20S shed light on their selectivity for mycobacterial over human proteasomes.

Mycobacterium tuberculosis cysteine biosynthesis genes mec+-cysO-cysM confer resistance to clofazimine.

The Mycobacterium tuberculosis mec+-cysO-cysM gene cluster was shown to be part of a novel cysteine biosynthesis pathway in vitro, but little is known about its essentiality or role in M. tuberculosis physiology. In this study, we generate a knock out of the mec+-cysO-cysM gene cluster in M. tuberculosis and show that the gene cluster is not essential under a variety of conditions, suggesting redundancy in pathways for cysteine biosynthesis in M. tuberculosis. The cysteine biosynthesis gene cluster is essential for resistance for clofazimine, a peroxide-producing anti-leprosy drug. Therefore, although under most conditions the pathway is not essential, it likely has an important role in defense against oxidative stress in M. tuberculosis.

Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

Steroid-Responsive Relapsing-Remitting Neutrophilic Encephalitis: A Case Report.

We report a case of a rapidly progressing, relapsing-remitting, steroid-responsive granulocytic encephalitis without any signs of peripheral nervous system or other organ involvement. It apparently had an immune-mediated etiology that could not be attributed to any known disease entity. A 22-year-old man presented with rapidly progressive severe neurological symptoms caused by encephalitis. Examination of the cerebrospinal fluid as well as brain biopsy showed extensive accumulation of neutrophilic granulocytes with no hints of an infectious agent. Magnetic resonance imaging revealed multiple T2/FLAIR demarcated lesions. Subsequent to a steroid pulse therapy, the clinical symptoms and imaging abnormalities improved rapidly. Ten months later, the patient experienced a disease relapse, which again responded well to steroids. Forty months after the relapse, he is currently doing well on azathioprine. This case highlights that an immunosuppressive treatment should be considered in patients with extensive neutrophilic encephalitis when no infectious agent is detected. A new immune-mediated relapsing-remitting CNS disease entity might need to be considered.

Diagnostic accuracy of DW MR imaging in the differentiation of chordomas and chondrosarcomas of the skull base: A 3.0-T MRI study of 105 cases.

PURPOSE: To determine the diagnostic accuracy of DW MR imaging with apparent diffusion coefficient (ADC) mapping for the distinction of skull base chordoma from skull base chondrosarcoma. PATIENTS AND METHODS: From October 2009 to April 2014, 105 consecutive patients (chordomas (n = 70), and chondrosarcomas (n = 35) who were planned to receive proton or heavy-ion beam therapy underwent DW MR imaging on a 3.0 T. MR images were assessed by means of consensus of three experienced radiologists who were blinded to pathologic and clinical information. ADC values (mean, minimum, maximum, and normalized) of the solid tumor component were evaluated. Group means and cut-off points were established to separate skull base chordoma from skull base chondrosarcoma, and statistical significances were calculated by Student's t or Mann-Whitney-U tests, and receiver operating characteristic (ROC) curve analyses. The prospective study was approved by the institutional ethics committee. RESULTS: For solid tumor component, there were higher mean, minimum, maximum, and normalized ADC values in chondrosarcomas compared with those in chordomas (all p < 0.001). ROC analysis revealed areas under the curve for mean, minimum, maximum, and normalized ADC values between 0.79 and 0.93. The mean ADC value of the solid tumor components had the best AUC, with a cut-off point of 1585 × 10(-6) mm (2)/s and sensitivity and specificity of 94.3% and 98.6%, respectively. CONCLUSION: Skull base Chondrosarcomas generally have higher mean, minimum, maximum, and normalized ADC values than skull base chordomas, with the mean ADC value of the solid tumor component offering the highest accuracy for characterization.

FGFR1:TACC1 fusion is a frequent event in molecularly defined extraventricular neurocytoma.

Extraventricular neurocytoma (EVN) is a rare primary brain tumor occurring in brain parenchyma outside the ventricular system. Histopathological characteristics resemble those of central neurocytoma but exhibit a wider morphologic spectrum. Accurate diagnosis of these histologically heterogeneous tumors is often challenging because of the overlapping morphological features and the lack of defining molecular markers. Here, we explored the molecular landscape of 40 tumors diagnosed histologically as EVN by investigating copy number profiles and DNA methylation array data. DNA methylation profiles were compared with those of relevant differential diagnoses of EVN and with a broader spectrum of diverse brain tumor entities. Based on this, our tumor cohort segregated into different groups. While a large fraction (n = 22) formed a separate epigenetic group clearly distinct from established DNA methylation profiles of other entities, a subset (n = 14) of histologically diagnosed EVN grouped with clusters of other defined entities. Three cases formed a small group close to but separated from the epigenetically distinct EVN cases, and one sample clustered with non-neoplastic brain tissue. Four additional samples originally diagnosed otherwise were found to molecularly resemble EVN. Thus, our results highlight a distinct DNA methylation pattern for the majority of tumors diagnosed as EVN, but also indicate that approximately one third of morphological diagnoses of EVN epigenetically correspond to other brain tumor entities. Copy number analysis and confirmation through RNA sequencing revealed FGFR1-TACC1 fusion as a distinctive, recurrent feature within the EVN methylation group (60%), in addition to a small number of other FGFR rearrangements (13%). In conclusion, our data demonstrate a specific epigenetic signature of EVN suitable for characterization of these tumors as a molecularly distinct entity, and reveal a high frequency of potentially druggable FGFR pathway activation in this tumor group.

Practical implementation of DNA methylation and copy-number-based CNS tumor diagnostics: the Heidelberg experience.

Recently, we described a machine learning approach for classification of central nervous system tumors based on the analysis of genome-wide DNA methylation patterns [6]. Here, we report on DNA methylation-based central nervous system (CNS) tumor diagnostics conducted in our institution between the years 2015 and 2018. In this period, more than 1000 tumors from the neurosurgical departments in Heidelberg and Mannheim and more than 1000 tumors referred from external institutions were subjected to DNA methylation analysis for diagnostic purposes. We describe our current approach to the integrated diagnosis of CNS tumors with a focus on constellations with conflicts between morphological and molecular genetic findings. We further describe the benefit of integrating DNA copy-number alterations into diagnostic considerations and provide a catalog of copy-number changes for individual DNA methylation classes. We also point to several pitfalls accompanying the diagnostic implementation of DNA methylation profiling and give practical suggestions for recurring diagnostic scenarios.

Integrated analysis of dynamic FET PET/CT parameters, histology, and methylation profiling of 44 gliomas.

PURPOSE: Dynamic 18F-FET PET/CT is a powerful tool for the diagnosis of gliomas.18F-FET PET time-activity curves (TAC) allow differentiation between histological low-grade gliomas (LGG) and high-grade gliomas (HGG). Molecular methods such as epigenetic profiling are of rising importance for glioma grading and subclassification. Here, we analysed dynamic 18F-FET PET data, and the histological and epigenetic features of 44 gliomas. METHODS: Dynamic 18F-FET PET was performed in 44 patients with newly diagnosed, untreated glioma: 10 WHO grade II glioma, 13 WHO grade III glioma and 21 glioblastoma (GBM). All patients underwent stereotactic biopsy or tumour resection after 18F-FET PET imaging. As well as histological analysis of tissue samples, DNA was subjected to epigenetic analysis using the Illumina 850 K methylation array. TACs, standardized uptake values corrected for background uptake in healthy tissue (SUVmax/BG), time to peak (TTP) and kinetic modelling parameters were correlated with histological diagnoses and with epigenetic signatures. Multivariate analyses were performed to evaluate the diagnostic accuracy of 18F-FET PET in relation to the tumour groups identified by histological and methylation-based analysis. RESULTS: Epigenetic profiling led to substantial tumour reclassification, with six grade II/III gliomas reclassified as GBM. Overlap of HGG-typical TACs and LGG-typical TACs was dramatically reduced when tumours were clustered on the basis of their methylation profile. SUVmax/BG values of GBM were higher than those of LGGs following both histological diagnosis and methylation-based diagnosis. The differences in TTP between GBMs and grade II/III gliomas were greater following methylation-based diagnosis than following histological diagnosis. Kinetic modeling showed that relative K1 and fractal dimension (FD) values significantly differed in histology- and methylation-based GBM and grade II/III glioma between those diagnosed histologically and those diagnosed by methylation analysis. Multivariate analysis revealed slightly greater diagnostic accuracy with methylation-based diagnosis. IDH-mutant gliomas and GBM subgroups tended to differ in their 18F-FET PET kinetics. CONCLUSION: The status of dynamic 18F-FET PET as a biologically and clinically relevant imaging modality is confirmed in the context of molecular glioma diagnosis.

Novel, improved grading system(s) for IDH-mutant astrocytic gliomas.

According to the 2016 World Health Organization Classification of Tumors of the Central Nervous System (2016 CNS WHO), IDH-mutant astrocytic gliomas comprised WHO grade II diffuse astrocytoma, IDH-mutant (AIIIDHmut), WHO grade III anaplastic astrocytoma, IDH-mutant (AAIIIIDHmut), and WHO grade IV glioblastoma, IDH-mutant (GBMIDHmut). Notably, IDH gene status has been made the major criterion for classification while the manner of grading has remained unchanged: it is based on histological criteria that arose from studies which antedated knowledge of the importance of IDH status in diffuse astrocytic tumor prognostic assessment. Several studies have now demonstrated that the anticipated differences in survival between the newly defined AIIIDHmut and AAIIIIDHmut have lost their significance. In contrast, GBMIDHmut still exhibits a significantly worse outcome than its lower grade IDH-mutant counterparts. To address the problem of establishing prognostically significant grading for IDH-mutant astrocytic gliomas in the IDH era, we undertook a comprehensive study that included assessment of histological and genetic approaches to prognosis in these tumors. A discovery cohort of 211 IDH-mutant astrocytic gliomas with an extended observation was subjected to histological review, image analysis, and DNA methylation studies. Tumor group-specific methylation profiles and copy number variation (CNV) profiles were established for all gliomas. Algorithms for automated CNV analysis were developed. All tumors exhibiting 1p/19q codeletion were excluded from the series. We developed algorithms for grading, based on molecular, morphological and clinical data. Performance of these algorithms was compared with that of WHO grading. Three independent cohorts of 108, 154 and 224 IDH-mutant astrocytic gliomas were used to validate this approach. In the discovery cohort several molecular and clinical parameters were of prognostic relevance. Most relevant for overall survival (OS) was CDKN2A/B homozygous deletion. Other parameters with major influence were necrosis and the total number of CNV. Proliferation as assessed by mitotic count, which is a key parameter in 2016 CNS WHO grading, was of only minor influence. Employing the parameters most relevant for OS in our discovery set, we developed two models for grading these tumors. These models performed significantly better than WHO grading in both the discovery and the validation sets. Our novel algorithms for grading IDH-mutant astrocytic gliomas overcome the challenges caused by introduction of IDH status into the WHO classification of diffuse astrocytic tumors. We propose that these revised approaches be used for grading of these tumors and incorporated into future WHO criteria.

Anaplastic astrocytoma with piloid features, a novel molecular class of IDH wildtype glioma with recurrent MAPK pathway, CDKN2A/B and ATRX alterations.

Tumors with histological features of pilocytic astrocytoma (PA), but with increased mitotic activity and additional high-grade features (particularly microvascular proliferation and palisading necrosis) have often been designated anaplastic pilocytic astrocytomas. The status of these tumors as a separate entity has not yet been conclusively demonstrated and molecular features have only been partially characterized. We performed DNA methylation profiling of 102 histologically defined anaplastic pilocytic astrocytomas. T-distributed stochastic neighbor-embedding (t-SNE) and hierarchical clustering analysis of these 102 cases against 158 reference cases from 12 glioma reference classes revealed that a subset of 83 of these tumors share a common DNA methylation profile that is distinct from the reference classes. These 83 tumors were thus denominated DNA methylation class anaplastic astrocytoma with piloid features (MC AAP). The 19 remaining tumors were distributed amongst the reference classes, with additional testing confirming the molecular diagnosis in most cases. Median age of patients with MC AAP was 41.5 years. The most frequent localization was the posterior fossa (74%). Deletions of CDKN2A/B (66/83, 80%), MAPK pathway gene alterations (49/65, 75%, most frequently affecting NF1, followed by BRAF and FGFR1) and mutations of ATRX or loss of ATRX expression (33/74, 45%) were the most common molecular alterations. All tumors were IDH1/2 wildtype. The MGMT promoter was methylated in 38/83 tumors (45%). Outcome analysis confirmed an unfavorable clinical course in comparison to PA, but better than IDH wildtype glioblastoma. In conclusion, we show that a subset of histologically defined anaplastic pilocytic astrocytomas forms a separate DNA methylation cluster, harbors recurrent alterations in MAPK pathway genes in combination with alterations of CDKN2A/B and ATRX, affects patients who are on average older than those diagnosed with PA and has an intermediate clinical outcome.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Targeting the Proteostasis Network for Mycobacterial Drug Discovery.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the world's deadliest infectious diseases and urgently requires new antibiotics to treat drug-resistant strains and to decrease the duration of therapy. During infection, Mtb encounters numerous stresses associated with host immunity, including hypoxia, reactive oxygen and nitrogen species, mild acidity, nutrient starvation, and metal sequestration and intoxication. The Mtb proteostasis network, composed of chaperones, proteases, and a eukaryotic-like proteasome, provides protection from stresses and chemistries of host immunity by maintaining the integrity of the mycobacterial proteome. In this Review, we explore the proteostasis network as a noncanonical target for antibacterial drug discovery.