ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL)1–3, but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia

SARS-CoV-2-specific immune responses and clinical outcomes after COVID-19 vaccination in patients with immune-suppressive disease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune responses and infection outcomes were evaluated in 2,686 patients with varying immune-suppressive disease states after administration of two Coronavirus Disease 2019 (COVID-19) vaccines. Overall, 255 of 2,204 (12%) patients failed to develop anti-spike antibodies, with an additional 600 of 2,204 (27%) patients generating low levels (<380 AU ml−1). Vaccine failure rates were highest in ANCA-associated vasculitis on rituximab (21/29, 72%), hemodialysis on immunosuppressive therapy (6/30, 20%) and solid organ transplant recipients (20/81, 25% and 141/458, 31%). SARS-CoV-2-specific T cell responses were detected in 513 of 580 (88%) patients, with lower T cell magnitude or proportion in hemodialysis, allogeneic hematopoietic stem cell transplantation and liver transplant recipients (versus healthy controls). Humoral responses against Omicron (BA.1) were reduced, although cross-reactive T cell responses were sustained in all participants for whom these data were available. BNT162b2 was associated with higher antibody but lower cellular responses compared to ChAdOx1 nCoV-19 vaccination. We report 474 SARS-CoV-2 infection episodes, including 48 individuals with hospitalization or death from COVID-19. Decreased magnitude of both the serological and the T cell response was associated with severe COVID-19. Overall, we identified clinical phenotypes that may benefit from targeted COVID-19 therapeutic strategies

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

Genetic basis of familial gastric cancer : beyond the e-cadherin (CDH1) locus

Importance. Familial aggregation occurs in approximately 10% of gastric cancers, which are generally sub-classified histologically as intestinal-type and diffuse gastric cancers. Though the genetic basis of familial intestinal-type gastric cancers is not known, in <50% of families clinically defined as hereditary diffuse gastric cancer, germline mutations in the E-cadherin gene, CDH1, are detected. This has lead to management guidelines and prevention strategies for mutation carriers. Objectives. To determine whether pathogenic germline mutations in genes alternative to CDH1 can be found in hereditary gastric cancer families using a multiplex panel sequencing approach. Design, Setting and Participants. One hundred fifteen probands from families who met the International Gastric Cancer Linkage Consortium clinical criteria for hereditary diffuse gastric cancer (n=106) or familial intestinal-type gastric cancer (n=9) were included. All diffuse gastric cancer probands tested negative for CDH1-mutations. Germline DNA was screened against a custom panel of 55 genes, including 14 prospective gastric cancer susceptibility genes, using a multiplexed amplicon-based next generation sequencing assay. Candidate mutations were validated via Sanger sequencing. Tumours from pathogenic mutation-positive probands were evaluated by immunohistochemistry. Results. Of 115 probands, four clearly pathogenic truncating mutations were identified in unrelated families, including two different mutations in CTNNA1 (alpha-catenin) and two different mutations in BRCA2. Previously described, functionally pathogenic missense mutations in SDHB (2 families) and STK11 (2 families) were also seen. Additional truncating mutations of likely lower penetrance were identified in ATM (4 families), MSR1 (2 families) and PALB2 (1 family). Cancers from carriers of CTNNA1 truncating variants had prominent loss of protein expression, further supporting their pathogenicity. Conclusion and Relevance. Using a multi-gene panel, families with hereditary gastric cancer were found to carry pathogenic mutations in genes commonly associated with other cancer susceptibility syndromes. In addition, this data suggests that familial gastric cancers, specifically hereditary diffuse gastric cancer syndrome, may benefit from a genetic, rather than clinical, classification. The genetic basis of the remaining families is likely attributable to mutations in genes yet to be implicated in hereditary gastric cancer or, in the diffuse gastric cancer families, atypical aberrations in the non-coding regions of CDH1.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Ixodes ricinus and Borrelia burgdorferi sensu lato in the Royal Parks of London, UK

Assessing the risk of tick-borne disease in areas with high visitor numbers is important from a public health perspective. Evidence suggests that tick presence, density, infection prevalence and the density of infected ticks can vary between habitats within urban green space, suggesting that the risk of Lyme borreliosis transmission can also vary. This study assessed nymph density, Borrelia prevalence and the density of infected nymphs across a range of habitat types in nine parks in London which receive millions of visitors each year. Ixodes ricinus were found in only two of the nine locations sampled, and here they were found in all types of habitat surveyed. Established I. ricinus populations were identified in the two largest parks, both of which had resident free-roaming deer populations. Highest densities of nymphs (15.68 per 100 m2) and infected nymphs (1.22 per 100 m2) were associated with woodland and under canopy habitats in Richmond Park, but ticks infected with Borrelia were found across all habitat types surveyed. Nymphs infected with Borrelia (7.9%) were only reported from Richmond Park, where Borrelia burgdorferi sensu stricto and Borrelia afzelii were identified as the dominant genospecies. Areas with short grass appeared to be less suitable for ticks and maintaining short grass in high footfall areas could be a good strategy for reducing the risk of Lyme borreliosis transmission to humans in such settings. In areas where this would create conflict with existing practices which aim to improve and/or meet historic landscape, biodiversity and public access goals, promoting public health awareness of tick-borne disease risks could also be utilised

Point mutations in Exon 1B of APC reveal gastric adenocarcinoma and proximal polyposis of the stomach as a familial adenomatous polyposis variant

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in\ua0vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present

EXTH-36. COMBINING ONC201 AND PAXALISIB FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA (DMG); THE PRECLINICAL RESULTS UNDERPINNING THE INTERNATIONAL PHASE II CLINICAL TRIAL (NCT05009992)

Diffuse midline gliomas (DMGs) diagnosed in the pons (DIPG) are universally fatal central nervous system tumors and are the leading cause of cancer-related death in children. Palliative radiotherapy is the only recognized treatment, with median overall survival just 9-11 months. The brain-penetrant, small molecule, ONC201, shows early-stage clinical trial efficacy, extending survival by ~9-11 months compared to historic controls. However, studies to determine the mechanisms behind the temporary clinical response to ONC201 are needed. Here, we have used a systems-biological approach to investigate whether genomic features influenced ONC201 response. DMGs harboring PIK3CA mutations were more sensitive to ONC201, whereas those harboring TP53 mutations were less sensitive. Quantitative proteogenomics identified that ONC201 elicits potent agonism of the mitochondrial protease, ClpP, driving proteolysis of electron transport chain and tricarboxylic acid proteins, leading to mitochondrial dysfunction. However, metabolic adaptation to ONC201 is promoted by the spare redox-signaling capacity of cells harboring WT-PIK3CA that was counteracted using the brain-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. ONC201 and paxalisib combinations extended survival of orthotopic DIPG xenograft mouse models (SU-DIPG-VI, p=0.0027; SF8626, p=0.0002; HSJD-DIPG-007, p=&amp;lt; 0.0001). The combination in the first three recorded patients; two at progression following re-irradiation, and one at diagnosis following the completion of radiation, resulted in dramatic reductions in tumor area, dramatically extending overall survival for all three patients (25 months, 30 and 31 months continuing). The DIPG patient receiving the combination since diagnosis, remains in progression free survival (MR axial diagnosis scan = 1554 mm2, current tumour area = 306 mm2, ~80% reduction). The patient continuing to receive the combination at progression and following reirradiation also experienced a marked decrease in tumor size (MR axial diagnosis scan = 1248 mm2, current tumour area = 315 mm2, ~75% reduction), 10 months following radiological detection of progression. These data inform the phase II clinical trial (NCT05009992)

Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern