Multidimensional genotyping of formalin-fixed paraffin-embedded (FFPE) samples has the potential to improve diagnostics and clinical trials for brain tumors, but prospective use in the clinical ...setting is not yet routine. We report our experience with implementing a multiplexed copy number and mutation-testing program in a diagnostic laboratory certified by the Clinical Laboratory Improvement Amendments.
We collected and analyzed clinical testing results from whole-genome array comparative genomic hybridization (OncoCopy) of 420 brain tumors, including 148 glioblastomas. Mass spectrometry-based mutation genotyping (OncoMap, 471 mutations) was performed on 86 glioblastomas.
OncoCopy was successful in 99% of samples for which sufficient DNA was obtained (n = 415). All clinically relevant loci for glioblastomas were detected, including amplifications (EGFR, PDGFRA, MET) and deletions (EGFRvIII, PTEN, 1p/19q). Glioblastoma patients ≤40 years old had distinct profiles compared with patients >40 years. OncoMap testing reliably identified mutations in IDH1, TP53, and PTEN. Seventy-seven glioblastoma patients enrolled on trials, of whom 51% participated in targeted therapeutic trials where multiplex data informed eligibility or outcomes. Data integration identified patients with complete tumor suppressor inactivation, albeit rarely (5% of patients) due to lack of whole-gene coverage in OncoMap.
Combined use of multiplexed copy number and mutation detection from FFPE samples in the clinical setting can efficiently replace singleton tests for clinical diagnosis and prognosis in most settings. Our results support incorporation of these assays into clinical trials as integral biomarkers and their potential to impact interpretation of results. Limited tumor suppressor variant capture by targeted genotyping highlights the need for whole-gene sequencing in glioblastoma.
Comprehensive multiplatform analysis of 80 uveal melanomas (UM) identifies four molecularly distinct, clinically relevant subtypes: two associated with poor-prognosis monosomy 3 (M3) and two with ...better-prognosis disomy 3 (D3). We show that BAP1 loss follows M3 occurrence and correlates with a global DNA methylation state that is distinct from D3-UM. Poor-prognosis M3-UM divide into subsets with divergent genomic aberrations, transcriptional features, and clinical outcomes. We report change-of-function SRSF2 mutations. Within D3-UM, EIF1AX- and SRSF2/SF3B1-mutant tumors have distinct somatic copy number alterations and DNA methylation profiles, providing insight into the biology of these low- versus intermediate-risk clinical mutation subtypes.
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•Both D3 and M3-UM divide into molecularly distinct subsets with different outcomes•Poor-prognosis M3-UM are characterized by a global DNA methylation pattern•Poor-prognosis M3-UM subsets have distinct genomic, signaling, and immune profiles•EIF1AX and SRSF2/SF3B1 mutant D3-UM have different genomic/DNA methylation profiles
Robertson et al. analyze 80 uveal melanomas (UM) and divide poor-prognosis monosomy 3 UM into subsets with divergent genomic aberrations, transcriptional features, and clinical outcomes. Somatic copy number changes and DNA methylation profiles separate better-prognosis disomy 3 UM into low or intermediate risk.
We report a comprehensive molecular characterization of pheochromocytomas and paragangliomas (PCCs/PGLs), a rare tumor type. Multi-platform integration revealed that PCCs/PGLs are driven by diverse ...alterations affecting multiple genes and pathways. Pathogenic germline mutations occurred in eight PCC/PGL susceptibility genes. We identified CSDE1 as a somatically mutated driver gene, complementing four known drivers (HRAS, RET, EPAS1, and NF1). We also discovered fusion genes in PCCs/PGLs, involving MAML3, BRAF, NGFR, and NF1. Integrated analysis classified PCCs/PGLs into four molecularly defined groups: a kinase signaling subtype, a pseudohypoxia subtype, a Wnt-altered subtype, driven by MAML3 and CSDE1, and a cortical admixture subtype. Correlates of metastatic PCCs/PGLs included the MAML3 fusion gene. This integrated molecular characterization provides a comprehensive foundation for developing PCC/PGL precision medicine.
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•Comprehensive molecular profiling of 173 pheochromocytoma and paraganglioma tumors•Single drivers in tumors by germline mutation, somatic mutation, or fusion gene•MAML3 fusion gene and CSDE1 somatic mutation define a Wnt-altered subtype•Prognostic markers of metastatic disease include the MAML3 fusion gene
Fishbein et al. show that neuroendocrine tumors, pheochromocytomas and paragangliomas, have a low genome alteration rate but diverse driver alterations, which coalesce into four molecular subtypes. The Wnt-altered subtype, driven by MAML3 fusions and CSDE1 somatic mutations, correlates with poor clinical outcome.
Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To ...understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 Å, 2.50 Å and 1.71 Å resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30° and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP γ phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 Å resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.
In contrast to RNA viruses, double-stranded DNA viruses have low mutation rates yet must still adapt rapidly in response to changing host defenses. To determine mechanisms of adaptation, we subjected ...the model poxvirus vaccinia to serial propagation in human cells, where its antihost factor K3L is maladapted against the antiviral protein kinase R (PKR). Viruses rapidly acquired higher fitness via recurrent K3L gene amplifications, incurring up to 7%–10% increases in genome size. These transient gene expansions were necessary and sufficient to counteract human PKR and facilitated the gain of an adaptive amino acid substitution in K3L that also defeats PKR. Subsequent reductions in gene amplifications offset the costs associated with larger genome size while retaining adaptive substitutions. Our discovery of viral “gene-accordions” explains how poxviruses can rapidly adapt to defeat different host defenses despite low mutation rates and reveals how classical Red Queen conflicts can progress through unrecognized intermediates.
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► Poxviruses rapidly adapt against host defenses via highly specific gene amplification ► Gains in vaccinia fitness occur within only a few serial passages in human cells ► Gene expansions precede and can facilitate adaptation via point mutation ► Gene “accordions” reveal new mode of virus adaptation in double-stranded DNA viruses
Despite low mutation rates, poxviruses evolve rapidly via gene amplifications, which facilitate the emergence of better-adapted versions of viral factors by increasing the sampling potential for mutations. Gene amplifications are only transient, revealing a gene “accordion” strategy for virus adaptation.
Summary
Background
Onychomycosis is a fungal disease that affects the fingernails and toenails and is predominantly caused by dermatophytes. VT‐1161 is a novel inhibitor of fungal CYP51 through the ...inhibition of lanosterol demethylase, and has demonstrated potent activity against Trichophyton rubrum and Trichophyton mentagrophytes.
Objectives
To evaluate the safety and efficacy of four dosing regimens of orally administered VT‐1161 compared with placebo in patients with moderate‐to‐severe distal and lateral subungual onychomycosis of the toenail.
Methods
This was a phase II, randomized, double‐blind, placebo‐controlled, multicentre study (ClinicalTrials.gov identifier NCT02267356). Patients aged 18–70 years (n = 259) who had 25–75% mycotic involvement were randomized to five treatment groups. They received 300 mg VT‐1161 as a 2‐week daily dose, followed by a once‐weekly dose for either 10 or 22 weeks, or 600 mg VT‐1161 as a 2‐week daily dose, followed by a once‐weekly dose for either 10 or 22 weeks. All treatments were followed by a nontreatment period of 36 weeks. A matching placebo arm was included.
Results
In the intent‐to‐treat population, at week 48 the complete cure rates were 0% in the placebo group and ranged from 32% to 42% in the VT‐1161 treatment groups (P < 0·001 vs. placebo). VT‐1161 was well tolerated, with no evidence of an adverse effect on liver function or QT intervals.
Conclusions
VT‐1161 treatment led to high nail clearance rates and a favourable safety profile. VT‐1161 exhibits characteristics that appear promising for the treatment of this chronic and difficult‐to‐treat condition and warrants further evaluation in larger studies.
What is already known about this topic?
Onychomycosis is a fungal disease that is chronic and difficult to treat.
Topical drugs are currently available but have limited effectiveness. Oral drugs, while effective, suffer from side‐effects including liver function abnormalities.
The majority of patients are elderly on polypharmacy and are at high risk of drug interactions.
An effective and safe oral drug with low potential for liver toxicity and drug interaction is highly desirable.
What does this study add?
This study presents the first large phase II study of a novel tetrazole antifungal, VT‐1161.
The study shows that VT‐1161 has an encouraging safety profile and is highly effective in treating even hard‐to-treat cases of onychomycosis.
The new tetrazole provides a lower overall drug load with an excellent safety and tolerability profile.
VT‐1161 is as effective as or numerically better than the current standard of care, terbinafine.
Linked Comment: Barbieri. Br J Dermatol 2021; 184:191.
Plain language summary available online
Recent determination of the cAMP response element-binding protein (CREB) basic leucine zipper (bZIP) consensus CRE crystal structure revealed key dimerization and DNA binding features that are ...conserved among members of the CREB/CREM/ATF-1 family of transcription factors. Dimerization appeared to be mediated by a Tyr307–Glu312interhelical hydrogen bond and a Glu319–Arg314electrostatic interaction. An unexpected hexahydrated Mg2+ion was centered above the CRE in the dimer cavity. In the present study, we related these features to CREB dimerization and DNA binding. A Y307F substitution reduced dimer stability and DNA binding affinity, whereas a Y307R mutation produced a stabilizing effect. Mutation of Glu319 to Ala or Lys attenuated dimerization and DNA binding. Mg2+ ions enhanced the binding affinity of wild-type CREB to the palindromic CRE by ∼20-fold but did not do so for divergent CREs. Similarly, mutation of Lys304, which mediates the CREB interaction with the hydrated Mg2+, blocked CREB binding to the palindromic but not the variant CRE sequences. The distinct binding characteristics of the K304A mutants to the consensus and variant CRE sequences indicate that CREB binding to these elements is differentially regulated by Mg2+ ions. We suggest that CREB binds the consensus and variant CRE sequences through fundamentally distinct mechanisms.
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