Circulating Tumour DNA Biomarkers Associated with Outcomes in Metastatic Prostate Cancer Treated with Lutetium-177-PSMA-617.

Background: Lutetium-177-prostate-specific membrane antigen- 617 (Lu-PSMA) is an effective therapy for metastatic castration-resistant prostate cancer (mCRPC). However, treatment responses are heterogeneous despite stringent positron emission tomography (PET)-based imaging selection criteria. Molecularly based biomarkers have potential to refine patient selection and optimise outcomes. Objective: To identify circulating tumour DNA (ctDNA) features associated with treatment outcomes for men treated with Lu-PSMA. Design setting and participants: ctDNA from men treated with Lu-PSMA in combination with idronoxil for progressive mCRPC were analysed using an 85-gene customised sequencing assay. ctDNA fractions, molecular profiles, and the presence of alterations in aggressive-variant prostate cancer (AVPC) genes were analysed at baseline, cycle 3 and at disease progression. Intervention: Men received Lu-PSMA with idronoxil every 6 wk for up to six cycles. Outcome measurements and statistical analysis: Baseline and exit PSMA and fluorodeoxyglucose PET/computed tomography (CT) imaging was conducted at baseline and study exit. Single-photon emission CT (SPECT) scans were performed 24 h after Lu-PSMA. Blood samples were collected at baseline,cycle 3 and at disease progression. Cox proportional-hazards models were used to assess associations and derive hazard ratios (HRs) and confidence intervals (CIs) for associations between molecular factors, imaging features, and clinical outcomes. Results and limitations: Sixty samples from 32 men were sequenced (32 at baseline, 24 at cycle 3, four from patients with disease progression); two samples (baseline, on-treatment) from one individual were excluded from analysis owing to poor quality of the baseline sequencing data. Alterations in AVPC genes were associated with shorter prostate-specific antigen (PSA) progression-free survival (PFS) and overall survival (OS) in univariate (HR 3.4, 95% CI 1.5-7.7; p = 0.0036; and HR 3.3, 95% CI 1.4-7.7; p = 0.0063, respectively) and multivariate analyses (HR 4.8, 95% CI 1.8-13; p = 0.0014; and HR 4.1, 95% CI 1.6-11; p = 0.004). Conclusions: ctDNA alterations in AVPC genes were associated with shorter PSA PFS and OS among men treated with Lu-PSMA and intermittent idronoxil. These candidate molecular biomarkers warrant further study to determine whether they have predictive value and potential to guide synergistic combination strategies to enhance outcomes for men treated with Lu-PSMA for mCRPC. Patient summary: Certain DNA/gene changes detected in the blood of men with advanced prostate cancer were associated with shorter benefit from lutetium PSMA, a targeted radioactive therapy. This information may be useful in determining which men may benefit most from this treatment, but additional research is needed.

Quantitative ctDNA Detection in Hepatoblastoma: Implications for Precision Medicine.

Hepatoblastoma is characterized by driver mutations in CTNNB1, making it an attractive biomarker for a liquid biopsy approach utilizing circulating tumor DNA (ctDNA). This prospective observational study sought to ascertain the feasibility of ctDNA detection in patients with hepatoblastoma and explore its associations with established clinical indicators and biomarkers, including serum Alpha-fetoprotein (AFP). We obtained 38 plasma samples and 17 tumor samples from 20 patients with hepatoblastoma. These samples were collected at various stages: 10 at initial diagnosis, 17 during neoadjuvant chemotherapy, 6 post-operatively, and 5 at disease recurrence. Utilizing a bespoke sequencing assay we developed called QUENCH, we identified single nucleotide variants and deletions in CTNNB1 ctDNA. Our study demonstrated the capability to quantitate ctDNA down to a variant allele frequency of 0.3%, achieving a sensitivity of 90% for patients at initial diagnosis, and a specificity of 100% at the patient level. Notably, ctDNA positivity correlated with tumor burden, and ctDNA levels exhibited associations with macroscopic residual disease and treatment response. Our findings provide evidence for the utility of quantitative ctDNA detection in hepatoblastoma management. Given the distinct detection targets, ctDNA and AFP-based stratification and monitoring approaches could synergize to enhance clinical decision-making. Further research is needed to elucidate the interplay between ctDNA and AFP and determine the optimal clinical applications for both methods in risk stratification and residual disease detection.

Hyper-IgE Syndrome due to an Elusive Novel Intronic Homozygous Variant in DOCK8.

Rare, biallelic loss-of-function mutations in DOCK8 result in a combined immune deficiency characterized by severe and recurrent cutaneous infections, eczema, allergies, and susceptibility to malignancy, as well as impaired humoral and cellular immunity and hyper-IgE. The advent of next-generation sequencing technologies has enabled the rapid molecular diagnosis of rare monogenic diseases, including inborn errors of immunity. These advances have resulted in the implementation of gene-guided treatments, such as hematopoietic stem cell transplant for DOCK8 deficiency. However, putative disease-causing variants revealed by next-generation sequencing need rigorous validation to demonstrate pathogenicity. Here, we report the eventual diagnosis of DOCK8 deficiency in a consanguineous family due to a novel homozygous intronic deletion variant that caused aberrant exon splicing and subsequent loss of expression of DOCK8 protein. Remarkably, the causative variant was not initially detected by clinical whole-genome sequencing but was subsequently identified and validated by combining advanced genomic analysis, RNA-seq, and flow cytometry. This case highlights the need to adopt multipronged confirmatory approaches to definitively solve complex genetic cases that result from variants outside protein-coding exons and conventional splice sites.

Molecular patterns in salivary duct carcinoma identify prognostic subgroups.

Salivary duct carcinoma (SDCa) is a rare cancer with high rate of metastases and poor survival despite aggressive multimodality treatment. This study analyzes the genetic changes in SDCa, their impact on cancer pathways, and evaluates whether molecular patterns can identify subgroups with distinct clinical characteristics and outcome. Clinicopathologic details and tissue samples from 66 patients (48 males, 18 females) treated between 1995 and 2018 were obtained from multiple institutions. Androgen receptor (AR) was assessed by immunohistochemistry, and the Illumina TruSight 170 gene panel was used for DNA sequencing. Male gender, lympho-vascular invasion, lymph node metastasis, and smoking were significant predictors of disease-free survival. AR was present in 79%. Frequently encountered alterations were mutations in TP53 (51%), PIK3CA (32%) and HRAS (22%), as well as amplifications of CDK4/6 (22%), ERBB2 (21%), MYC (16%), and deletions of CDKN2A (13%). TP53 mutation and MYC amplifications were associated with decreased disease-free survival. Analysis of cancer pathways revealed that the PI3K pathway was most commonly affected. Alterations in the cell cycle pathway were associated with impaired disease-free survival (HR 2.6, P = 0.038). Three subgroups based on AR and ERBB2 status were identified, which featured distinct molecular patterns and outcome. Among AR positive SDCa, HRAS mutations were restricted to AR positive tumors without ERBB2 amplification and HRAS mutations featured high co-occurrence with PIK3CA alterations, which seems specific to SDCa. AR negative SDCa were associated with poor disease-free survival in multivariate analysis (HR 4.5, P = 0.010) and none of these tumors exhibited ERBB2 amplification or HRAS mutations. AR and ERBB2 status in SDCa thus classifies tumors with distinct molecular profiles relevant to future targeted therapy. Furthermore, clinical factors such as smoking and molecular features such as MYC amplification may serve as markers of poor prognosis of SDCa.

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

Molecular Portraits of Early Rheumatoid Arthritis Identify Clinical and Treatment Response Phenotypes.

There is a current imperative to unravel the hierarchy of molecular pathways that drive the transition of early to established disease in rheumatoid arthritis (RA). Herein, we report a comprehensive RNA sequencing analysis of the molecular pathways that drive early RA progression in the disease tissue (synovium), comparing matched peripheral blood RNA-seq in a large cohort of early treatment-naive patients, namely, the Pathobiology of Early Arthritis Cohort (PEAC). We developed a data exploration website (https://peac.hpc.qmul.ac.uk/) to dissect gene signatures across synovial and blood compartments, integrated with deep phenotypic profiling. We identified transcriptional subgroups in synovium linked to three distinct pathotypes: fibroblastic pauci-immune pathotype, macrophage-rich diffuse-myeloid pathotype, and a lympho-myeloid pathotype characterized by infiltration of lymphocytes and myeloid cells. This is suggestive of divergent pathogenic pathways or activation disease states. Pro-myeloid inflammatory synovial gene signatures correlated with clinical response to initial drug therapy, whereas plasma cell genes identified a poor prognosis subgroup with progressive structural damage.

IRF2 transcriptionally induces GSDMD expression for pyroptosis.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1ß (IL-1ß) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1ß secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.

Intrinsic apoptosis shapes the tumor spectrum linked to inactivation of the deubiquitinase BAP1.

Malignancies arising from mutation of tumor suppressors have unexplained tissue proclivity. For example, BAP1 encodes a widely expressed deubiquitinase for histone H2A, but germline mutations are predominantly associated with uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver, and pancreatic tissue but not in melanocytes and mesothelial cells. Ubiquitin ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing expression of the prosurvival genes Bcl2 and Mcl1. In contrast, BAP1 loss in melanocytes had little impact on expression of prosurvival genes, instead inducing Mitf Thus, BAP1 appears to modulate gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program.

The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification.

Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.

Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain.

The E3 ubiquitin ligase CRL4COP1/DET1 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with constitutive photomorphogenesis 1 (Cop1) deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes Vimentin and Gfap was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including Olig1, Olig2, and Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several Cop1-deficient mice also lacking c-Jun and Etv5, or lacking Etv5 and heterozygous for Etv1, were viable.

ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.

Non-canonical reader modules of BAZ1A promote recovery from DNA damage.

Members of the ISWI family of chromatin remodelers mobilize nucleosomes to control DNA accessibility and, in some cases, are required for recovery from DNA damage. However, it remains poorly understood how the non-catalytic ISWI subunits BAZ1A and BAZ1B might contact chromatin to direct the ATPase SMARCA5. Here, we find that the plant homeodomain of BAZ1A, but not that of BAZ1B, has the unusual function of binding DNA. Furthermore, the BAZ1A bromodomain has a non-canonical gatekeeper residue and binds relatively weakly to acetylated histone peptides. Using CRISPR-Cas9-mediated genome editing we find that BAZ1A and BAZ1B each recruit SMARCA5 to sites of damaged chromatin and promote survival. Genetic engineering of structure-designed bromodomain and plant homeodomain mutants reveals that reader modules of BAZ1A and BAZ1B, even when non-standard, are critical for DNA damage recovery in part by regulating ISWI factors loading at DNA lesions and supporting transcriptional programs required for survival.ISWI chromatin remodelers regulate DNA accessibility and have been implicated in DNA damage repair. Here, the authors uncover functions, in response to DNA damage, for the bromodomain of the ISWI subunit BAZ1B and for the non-canonical PHD and bromodomain modules of the paralog BAZ1A.

Transcription factor Etv5 is essential for the maintenance of alveolar type II cells.

Alveolar type II (AT2) cell dysfunction contributes to a number of significant human pathologies including respiratory distress syndrome, lung adenocarcinoma, and debilitating fibrotic diseases, but the critical transcription factors that maintain AT2 cell identity are unknown. Here we show that the E26 transformation-specific (ETS) family transcription factor Etv5 is essential to maintain AT2 cell identity. Deletion of Etv5 from AT2 cells produced gene and protein signatures characteristic of differentiated alveolar type I (AT1) cells. Consistent with a defect in the AT2 stem cell population, Etv5 deficiency markedly reduced recovery following bleomycin-induced lung injury. Lung tumorigenesis driven by mutant KrasG12D was also compromised by Etv5 deficiency. ERK activation downstream of Ras was found to stabilize Etv5 through inactivation of the cullin-RING ubiquitin ligase CRL4COP1/DET1 that targets Etv5 for proteasomal degradation. These findings identify Etv5 as a critical output of Ras signaling in AT2 cells, contributing to both lung homeostasis and tumor initiation.

ß-Cell Insulin Secretion Requires the Ubiquitin Ligase COP1.

A variety of signals finely tune insulin secretion by pancreatic ß cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in ß cells is critical for insulin secretion. Mice lacking COP1 in ß cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human ß-cell pathophysiology. In normal ß cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics.

INTRODUCTION: Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood. METHODS: We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy. RESULTS: We documented evidence for four major phenotypes of RA synovium - lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004). CONCLUSIONS: These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases. TRIAL REGISTRATION: ClinicalTrials.gov NCT01119859

Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.

Asthma is canonically thought of as a disorder of excessive Th2-driven inflammation in the airway, although recent studies have described heterogeneity with respect to asthma pathophysiology. We have previously described distinct phenotypes of asthma based on the presence or absence of a three-gene "Th2 signature" in bronchial epithelium, which differ in terms of eosinophilic inflammation, mucin composition, subepithelial fibrosis, and corticosteroid responsiveness. In the present analysis, we sought to describe Th2 inflammation in human asthmatic airways quantitatively with respect to known mediators of inflammation and intercellular communication. Using whole-genome microarray and quantitative real-time PCR analysis of endobronchial biopsies from 27 mild-to-moderate asthmatics and 13 healthy controls with associated clinical and demographic data, we found that asthmatic Th2 inflammation is expressed over a variable continuum, correlating significantly with local and systemic measures of allergy and eosinophilia. We evaluated a composite metric describing 79 coexpressed genes associated with Th2 inflammation against the biological space comprising cytokines, chemokines, and growth factors, identifying distinctive patterns of inflammatory mediators as well as Wnt, TGF-ß, and platelet-derived growth factor family members. This integrated description of the factors regulating inflammation, cell migration, and tissue remodeling in asthmatic airways has important consequences for the pathophysiological and clinical impacts of emerging asthma therapeutics targeting Th2 inflammation.