Host and environmental factors shape upper airway microbiota and respiratory health across the human lifespan.

Our understanding of the normal variation in the upper respiratory tract (URT) microbiota across the human lifespan and how these relate to host, environment, and health is limited. We studied the microbiota of 3,104 saliva (<10 year-olds)/oropharynx (≥10 year-olds) and 2,485 nasopharynx samples of 3,160 Dutch individuals 0-87 years of age, participating in a cross-sectional population-wide study (PIENTER-3) using 16S-rRNA sequencing. The microbiota composition was strongly related to age, especially in the nasopharynx, with maturation occurring throughout childhood and adolescence. Clear niche- and age-specific associations were found between the microbiota composition and host/environmental factors and health outcomes. Among others, social interaction, sex, and season were associated with the nasopharyngeal microbial community. By contrast, the oral microbiota was more related to antibiotics, tobacco, and alcohol use. We present an atlas of the URT microbiota across the lifespan in association with environment and health, establishing a baseline for future research.

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Modeling recent human evolution in mice by expression of a selected EDAR variant.

An adaptive variant of the human Ectodysplasin receptor, EDARV370A, is one of the strongest candidates of recent positive selection from genome-wide scans. We have modeled EDAR370A in mice and characterized its phenotype and evolutionary origins in humans. Our computational analysis suggests the allele arose in central China approximately 30,000 years ago. Although EDAR370A has been associated with increased scalp hair thickness and changed tooth morphology in humans, its direct biological significance and potential adaptive role remain unclear. We generated a knockin mouse model and find that, as in humans, hair thickness is increased in EDAR370A mice. We identify new biological targets affected by the mutation, including mammary and eccrine glands. Building on these results, we find that EDAR370A is associated with an increased number of active eccrine glands in the Han Chinese. This interdisciplinary approach yields unique insight into the generation of adaptive variation among modern humans.

Bulevirtide Combined with Pegylated Interferon for Chronic Hepatitis D.

BACKGROUND: In a phase 3 trial, bulevirtide monotherapy led to a virologic response in patients with chronic hepatitis D. Pegylated interferon (peginterferon) alfa-2a is recommended by guidelines as an off-label treatment for this disease. The role of combination therapy with bulevirtide and peginterferon alfa-2a, particularly with regard to finite treatment, is unclear. METHODS: In this phase 2b, open-label trial, we randomly assigned patients to receive peginterferon alfa-2a alone (180 µg per week) for 48 weeks; bulevirtide at a daily dose of 2 mg or 10 mg plus peginterferon alfa-2a (180 µg per week) for 48 weeks, followed by the same daily dose of bulevirtide for 48 weeks; or bulevirtide at a daily dose of 10 mg alone for 96 weeks. All the patients were followed for 48 weeks after the end of treatment. The primary end point was an undetectable level of hepatitis D virus (HDV) RNA at 24 weeks after the end of treatment. The primary comparison was between the 10-mg bulevirtide plus peginterferon alfa-2a group and the 10-mg bulevirtide monotherapy group. RESULTS: A total of 24 patients received peginterferon alfa-2a alone, 50 received 2 mg and 50 received 10 mg of bulevirtide plus peginterferon alfa-2a, and 50 received 10 mg of bulevirtide monotherapy. At 24 weeks after the end of treatment, HDV RNA was undetectable in 17% of the patients in the peginterferon alfa-2a group, in 32% of those in the 2-mg bulevirtide plus peginterferon alfa-2a group, in 46% of those in the 10-mg bulevirtide plus peginterferon alfa-2a group, and in 12% of those in the 10-mg bulevirtide group. For the primary comparison, the between-group difference was 34 percentage points (95% confidence interval, 15 to 50; P<0.001). At 48 weeks after the end of treatment, HDV RNA was undetectable in 25% of the patients in the peginterferon alfa-2a group, in 26% of those in the 2-mg bulevirtide plus peginterferon alfa-2a group, in 46% of those in the 10-mg bulevirtide plus peginterferon alfa-2a group, and in 12% of those in the 10-mg bulevirtide group. The most frequent adverse events were leukopenia, neutropenia, and thrombocytopenia. The majority of adverse events were of grade 1 or 2 in severity. CONCLUSIONS: The combination of 10-mg bulevirtide plus peginterferon alfa-2a was superior to bulevirtide monotherapy with regard to an undetectable HDV RNA level at 24 weeks after the end of treatment. (Funded by Gilead Sciences; MYR 204 ClinicalTrials.gov number, NCT03852433.).

A neuropathy-associated kinesin KIF1A mutation hyper-stabilizes the motor-neck interaction during the ATPase cycle.

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the ß7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on ß7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.

Glucoselysine, a unique advanced glycation end-product of the polyol pathway and its association with vascular complications in type 2 diabetes.

Glucoselysine (GL) is an unique advanced glycation end-product derived from fructose. The main source of fructose in vivo is the polyol pathway, and an increase in its activity leads to diabetic complications. Here, we aimed to demonstrate that GL can serve as an indicator of the polyol pathway activity. Additionally, we propose a novel approach for detecting GL in peripheral blood samples using liquid chromatography-tandem mass spectrometry and evaluate its clinical usefulness. We successfully circumvent interference from fructoselysine, which shares the same molecular weight as GL, by performing ultrafiltration and hydrolysis without reduction, successfully generating adequate peaks for quantification in serum. Furthermore, using immortalized aldose reductase KO mouse Schwann cells, we demonstrate that GL reflects the downstream activity of the polyol pathway and that GL produced intracellularly is released into the extracellular space. Clinical studies reveal that GL levels in patients with type 2 diabetes are significantly higher than those in healthy participants, while Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1) levels are significantly lower. Both GL and MG-H1 show higher values among patients with vascular complications; however, GL varies more markedly than MG-H1 as well as hemoglobin A1c, fasting plasma glucose, and estimated glomerular filtration rate. Furthermore, GL remains consistently stable under various existing drug treatments for type 2 diabetes, whereas MG-H1 is impacted. To the best of our knowledge, we provide important insights in predicting diabetic complications caused by enhanced polyol pathway activity via assessment of GL levels in peripheral blood samples from patients.

Efficacy and Findings of a Blinded Randomized Reintroduction Phase for the Low FODMAP Diet in Irritable Bowel Syndrome.

BACKGROUND & AIMS: The efficacy of a low fermentable oligo-, di-, monosaccharides and polyols (FODMAP) diet in irritable bowel syndrome (IBS) is well established. After the elimination period, a reintroduction phase aims to identify triggers. We studied the impact of a blinded reintroduction using FODMAP powders to objectively identify triggers and evaluated the effect on symptoms, quality of life, and psychosocial comorbidities. METHODS: Responders to a 6-week low FODMAP diet, defined by a drop in IBS symptom severity score (IBS-SSS) compared with baseline, entered a 9-week blinded randomized reintroduction phase with 6 FODMAP powders (fructans, fructose, galacto-oligosaccharides, lactose, mannitol, sorbitol) or control (glucose). A rise in IBS-SSS (≥50 points) defined a FODMAP trigger. Patients completed daily symptom diaries and questionnaires for quality of life and psychosocial comorbidities. RESULTS: In 117 recruited patients with IBS, IBS-SSS improved significantly after the elimination period compared with baseline (150 ± 116 vs 301 ± 97, P < .0001, 80% responders). Symptom recurrence was triggered in 85% of the FODMAP powders, by an average of 2.5 ± 2 FODMAPs/patient. The most prevalent triggers were fructans (56%) and mannitol (54%), followed by galacto-oligosaccharides, lactose, fructose, sorbitol, and glucose (respectively 35%, 28%, 27%, 23%, and 26%) with a significant increase in abdominal pain at day 1 for sorbitol/mannitol, day 2 for fructans/galacto-oligosaccharides, and day 3 for lactose. CONCLUSION: We confirmed the significant benefit of the low FODMAP diet in tertiary-care IBS. A blinded reintroduction revealed a personalized pattern of symptom recurrence, with fructans and mannitol as the most prevalent, and allows the most objective identification of individual FODMAP triggers. Ethical commission University hospital of Leuven reference number: s63629; Clinicaltrials.gov number: NCT04373304.

Within-host evolutionary dynamics and tissue compartmentalization during acute SARS-CoV-2 infection.

The global evolution of SARS-CoV-2 depends in part upon the evolutionary dynamics within individual hosts with varying immune histories. To characterize the within-host evolution of acute SARS-CoV-2 infection, we sequenced saliva and nasal samples collected daily from vaccinated and unvaccinated individuals early during infection. We show that longitudinal sampling facilitates high-confidence genetic variant detection and reveals evolutionary dynamics missed by less-frequent sampling strategies. Within-host dynamics in both unvaccinated and vaccinated individuals appeared largely stochastic; however, in rare cases, minor genetic variants emerged to frequencies sufficient for forward transmission. Finally, we detected significant genetic compartmentalization of viral variants between saliva and nasal swab sample sites in many individuals. Altogether, these data provide a high-resolution profile of within-host SARS-CoV-2 evolutionary dynamics.IMPORTANCEWe detail the within-host evolutionary dynamics of SARS-CoV-2 during acute infection in 31 individuals using daily longitudinal sampling. We characterized patterns of mutational accumulation for unvaccinated and vaccinated individuals, and observed that temporal variant dynamics in both groups were largely stochastic. Comparison of paired nasal and saliva samples also revealed significant genetic compartmentalization between tissue environments in multiple individuals. Our results demonstrate how selection, genetic drift, and spatial compartmentalization all play important roles in shaping the within-host evolution of SARS-CoV-2 populations during acute infection.

Spatial Atlas for Mapping Vascular Microcalcification Using 18F-NaF PET/CT: Application in Hyperphosphatemic Familial Tumoral Calcinosis.

BACKGROUND: Vascular calcification causes significant morbidity and occurs frequently in diseases of calcium/phosphate imbalance. Radiolabeled sodium fluoride positron emission tomography/computed tomography has emerged as a sensitive and specific method for detecting and quantifying active microcalcifications. We developed a novel technique to quantify and map total vasculature microcalcification to a common space, allowing simultaneous assessment of global disease burden and precise tracking of site-specific microcalcifications across time and individuals. METHODS: To develop this technique, 4 patients with hyperphosphatemic familial tumoral calcinosis, a monogenic disorder of FGF23 (fibroblast growth factor-23) deficiency with a high prevalence of vascular calcification, underwent radiolabeled sodium fluoride positron emission tomography/computed tomography imaging. One patient received serial imaging 1 year after treatment with an IL-1 (interleukin-1) antagonist. A radiolabeled sodium fluoride-based microcalcification score, as well as calcification volume, was computed at all perpendicular slices, which were then mapped onto a standardized vascular atlas. Segment-wise mCSmean and mCSmax were computed to compare microcalcification score levels at predefined vascular segments within subjects. RESULTS: Patients with hyperphosphatemic familial tumoral calcinosis had notable peaks in microcalcification score near the aortic bifurcation and distal femoral arteries, compared with a control subject who had uniform distribution of vascular radiolabeled sodium fluoride uptake. This technique also identified microcalcification in a 17-year-old patient, who had no computed tomography-defined calcification. This technique could not only detect a decrease in microcalcification score throughout the patient treated with an IL-1 antagonist but it also identified anatomic areas that had increased responsiveness while there was no change in computed tomography-defined macrocalcification after treatment. CONCLUSIONS: This technique affords the ability to visualize spatial patterns of the active microcalcification process in the peripheral vasculature. Further, this technique affords the ability to track microcalcifications at precise locations not only across time but also across subjects. This technique is readily adaptable to other diseases of vascular calcification and may represent a significant advance in the field of vascular biology.

Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009 to 2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome sequencing and WGS and, latterly, WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. In summary, 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100 000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment, leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre and has benefitted from the use of WGS, particularly access to the raw data. However, almost one-quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'.

Anatomo-functional basis of emotional and motor resonance elicited by facial expressions.

Simulation theories predict that the observation of other's expressions modulates neural activity in the same centres controlling their production. This hypothesis has been developed by two models, postulating that the visual input is directly projected either to the motor system for action recognition (motor resonance) or to emotional/interoceptive regions for emotional contagion and social synchronization (emotional resonance). Here we investigated the role of frontal/insular regions in the processing of observed emotional expressions by combining intracranial recording, electrical stimulation and effective connectivity. First, we intracranially recorded from prefrontal, premotor or anterior insular regions of 44 patients during the passive observation of emotional expressions, finding widespread modulations in prefrontal/insular regions (anterior cingulate cortex, anterior insula, orbitofrontal cortex and inferior frontal gyrus) and motor territories (Rolandic operculum and inferior frontal junction). Subsequently, we electrically stimulated the activated sites, finding that (i) in the anterior cingulate cortex and anterior insula, the stimulation elicited emotional/interoceptive responses, as predicted by the 'emotional resonance model'; (ii) in the Rolandic operculum it evoked face/mouth sensorimotor responses, in line with the 'motor resonance' model; and (iii) all other regions were unresponsive or revealed functions unrelated to the processing of facial expressions. Finally, we traced the effective connectivity to sketch a network-level description of these regions, finding that the anterior cingulate cortex and the anterior insula are reciprocally interconnected while the Rolandic operculum is part of the parieto-frontal circuits and poorly connected with the former. These results support the hypothesis that the pathways hypothesized by the 'emotional resonance' and the 'motor resonance' models work in parallel, differing in terms of spatio-temporal fingerprints, reactivity to electrical stimulation and connectivity patterns.

Intravascular ultrasound-guided drug-coated balloon angioplasty for femoropopliteal artery disease: a clinical trial.

BACKGROUND AND AIMS: Drug-coated balloons (DCBs) have demonstrated favourable outcomes following endovascular therapy for femoropopliteal artery (FPA) disease. However, uncertainty remains whether the use of intravascular ultrasound (IVUS) can improve the outcomes of DCBs. METHODS: This prospective, multicentre, randomized trial, conducted at seven centres in South Korea, compared the outcomes of IVUS-guided vs. angiography-guided angioplasty for treating FPA disease with DCBs. Patients were assigned to receive IVUS-guided (n = 119) or angiography-guided (n = 118) angioplasty using DCBs. The primary endpoint was 12-month primary patency. RESULTS: Between May 2016 and August 2022, 237 patients were enrolled and 204 (86.0%) completed the trial (median follow-up; 363 days). The IVUS guidance group showed significantly higher primary patency [83.8% vs. 70.1%; cumulative difference 19.6% (95% confidence interval 6.8 to 32.3); P = .01] and increased freedom from clinically driven target lesion revascularization [92.4% vs. 83.0%; difference 11.6% (95% confidence interval 3.1 to 20.1); P = .02], sustained clinical improvement (89.1% vs. 76.3%, P = .01), and haemodynamic improvement (82.4% vs. 66.9%, P = .01) at 12 months compared with the angiography guidance group. The IVUS group utilized larger balloon diameters and pressures for pre-dilation, more frequent post-dilation, and higher pressures for post-dilation, resulting in a greater post-procedural minimum lumen diameter (3.90 ± 0.59 vs. 3.71 ± 0.73 mm, P = .03). CONCLUSIONS: Intravascular ultrasound guidance significantly improved the outcomes of DCBs for FPA disease in terms of primary patency, freedom from clinically driven target lesion revascularization, and sustained clinical and haemodynamic improvement at 12 months. These benefits may be attributed to IVUS-guided optimization of the lesion before and after DCB treatment.

PEGylated liposomes for diagnosis of polyethylene glycol allergy.

BACKGROUND: Polyethylene glycol (PEG) is a nonprotein polymer that is present in its native (unbound) form as an excipient in a range of products. It is increasingly being utilized clinically in the form of PEGylated liposomal medications and vaccines. PEG is the cause of anaphylaxis in a small percentage of drug reactions; however, diagnosis of PEG allergy is complicated by the variable and poor diagnostic performance of current skin testing protocols. OBJECTIVE: We assessed the diagnostic performance of PEGylated lipid medications as an alternative to currently described tests that use medications containing PEG excipients. METHODS: Nine patients with a strong history of PEG allergy were evaluated by skin testing with a panel of PEG-containing medications and with a PEGylated lipid nanoparticle vaccine (BNT162b2). Reactivity of basophils to unbound and liposomal PEG was assessed ex vivo, and specificity of basophil responses to PEGylated liposomes was investigated with a competitive inhibition assay. More detailed information is provided in this article's Methods section in the Online Repository available at www.jacionline.org. RESULTS: Despite compelling histories of anaphylaxis to PEG-containing medications, only 2 (22%) of 9 patients were skin test positive for purified PEG or their index reaction-indicated PEG-containing compound. Conversely, all 9 patients were skin test positive or basophil activation test positive to PEGylated liposomal BNT162b2 vaccine. Concordantly, PEGylated liposomal drugs (BNT162b2 vaccine and PEGylated liposomal doxorubicin), but not purified PEG2000, consistently induced basophil activation ex vivo in patients with PEG allergy but not in nonallergic controls. Basophil reactivity to PEGylated nanoparticles competitively inhibited by preincubation of basophils with native PEG2000. CONCLUSION: Presentation of PEG on the surface of a lipid nanoparticle increases its in vivo and ex vivo allergenicity, and improves diagnosis of PEG allergy.

Kinase fusion positive intra-osseous spindle cell tumors: A series of eight cases with review of the literature.

Mesenchymal spindle cell tumors with kinase fusions, often presenting in superficial or deep soft tissue locations, may rarely occur in bone. Herein, we describe the clinicopathologic and molecular data of eight bone tumors characterized by various kinase fusions from our files and incorporate the findings with the previously reported seven cases, mainly as single case reports. In the current series all but one of the patients were young children or teenagers, with an age range from newborn to 59 years (mean 19 years). Most tumors (n = 5) presented in the head and neck area (skull base, mastoid, maxilla, and mandible), and remaining three in the tibia, pelvic bone, and chest wall. The fusions included NTRK1 (n = 3), RET (n = 2), NTRK3 (n = 2), and BRAF (n = 1). In the combined series (n = 15), most tumors (73%) occurred in children and young adults (<30 years) and showed a predilection for jaw and skull bones (40%), followed by long and small tubular bones (33%). The fusions spanned a large spectrum of kinase genes, including in descending order NTRK3 (n = 6), NTRK1 (n = 4), RET (n = 2), BRAF (n = 2), and RAF1 (n = 1). All fusions confirmed by targeted RNA sequencing were in-frame and retained the kinase domain within the fusion oncoprotein. Similar to the soft tissue counterparts, most NTRK3-positive bone tumors in this series showed high-grade morphology (5/6), whereas the majority of NTRK1 tumors were low-grade (3/4). Notably, all four tumors presenting in the elderly were high-grade spindle cell sarcomas, with adult fibrosarcoma (FS)-like, malignant peripheral nerve sheath tumor (MPNST)-like and MPNST phenotypes. Overall, 10 tumors had high-grade morphology, ranging from infantile and adult-types FS, MPNST-like, and MPNST, whereas five showed benign/low-grade histology (MPNST-like and myxoma-like). Immunohistochemically (IHC), S100 and CD34 positivity was noted in 57% and 50%, respectively, while co-expression of S100 and CD34 in 43% of cases. One-third of tumors (4 high grade and the myxoma-like) were negative for both S100 and CD34. IHC for Pan-TRK was positive in all eight NTRK-fusion positive tumors tested and negative in two tumors with other kinase fusions. Clinical follow-up was too limited to allow general conclusions.

Recombinant Full-length Plasmodium falciparum Circumsporozoite Protein-Based Vaccine Adjuvanted With Glucopyranosyl Lipid A-Liposome Quillaja saponaria 21: Results of Phase 1 Testing With Malaria Challenge.

BACKGROUND: Malaria is preventable yet causes >600 000 deaths annually. RTS,S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. METHODS: We conducted an open-label, dose escalation phase 1 study of a full-length recombinant circumsporozoite protein vaccine (rCSP) administered with adjuvant glucopyranosyl lipid A-liposome Quillaja saponaria 21 formulation (GLA-LSQ) on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naive adults. The primary end points were safety and reactogenicity. The secondary end points were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection. RESULTS: Participants were enrolled into 4 groups receiving rCSP/GLA-LSQ: 10 µg × 3 (n = 20), 30 µg × 3 (n = 10), 60 µg × 3 (n = 10), or 60 µg × 2 (n = 9); 10 participants received 30 µg rCSP alone × 3, and there were 6 infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent controlled human malaria infection 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher immunoglobulin G titers but did not achieve previously established RTS,S benchmarks. CONCLUSIONS: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess whether adjuvant or schedule adjustments improve efficacy. CLINICAL TRIALS REGISTRATION: NCT03589794.

HBcrAg values may predict virological and immunological responses to pegIFN-α in NUC-suppressed HBeAg-negative chronic hepatitis B.

OBJECTIVE: Selected populations of patients with chronic hepatitis B (CHB) may benefit from a combined use of pegylated interferon-alpha (pegIFN-α) and nucleos(t)ides (NUCs). The aim of our study was to assess the immunomodulatory effect of pegIFN-α on T and natural killer (NK) cell responses in NUC-suppressed patients to identify cellular and/or serological parameters to predict better T cell-restoring effect and better control of infection in response to pegIFN-α for a tailored application of IFN-α add-on. DESIGN: 53 HBeAg-negative NUC-treated patients with CHB were randomised at a 1:1 ratio to receive pegIFN-α-2a for 48 weeks, or to continue NUC therapy and then followed up for at least 6 months maintaining NUCs. Serum hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) levels as well as peripheral blood NK cell phenotype and function and HBV-specific T cell responses upon in vitro stimulation with overlapping HBV peptides were measured longitudinally before, during and after pegIFN-α therapy. RESULTS: Two cohorts of pegIFN-α treated patients were identified according to HBsAg decline greater or less than 0.5 log at week 24 post-treatment. PegIFN-α add-on did not significantly improve HBV-specific T cell responses during therapy but elicited a significant multispecific and polyfunctional T cell improvement at week 24 post-pegIFN-α treatment compared with baseline. This improvement was maximal in patients who had a higher drop in serum HBsAg levels and a lower basal HBcrAg values. CONCLUSIONS: PegIFN-α treatment can induce greater functional T cell improvement and HBsAg decline in patients with lower baseline HBcrAg levels. Thus, HBcrAg may represent an easily and reliably applicable parameter to select patients who are more likely to achieve better response to pegIFN-α add-on to virally suppressed patients.

Pembrolizumab plus concurrent chemoradiotherapy versus placebo plus concurrent chemoradiotherapy in patients with locally advanced squamous cell carcinoma of the head and neck (KEYNOTE-412): a randomised, double-blind, phase 3 trial.

BACKGROUND: Despite multimodal therapy, 5-year overall survival for locally advanced head and neck squamous cell carcinoma (HNSCC) is about 50%. We assessed the addition of pembrolizumab to concurrent chemoradiotherapy for locally advanced HNSCC. METHODS: In the randomised, double-blind, phase 3 KEYNOTE-412 trial, participants with newly diagnosed, high-risk, unresected locally advanced HNSCC from 130 medical centres globally were randomly assigned (1:1) to pembrolizumab (200 mg) plus chemoradiotherapy or placebo plus chemoradiotherapy. Randomisation was done using an interactive response technology system and was stratified by investigator's choice of radiotherapy regimen, tumour site and p16 status, and disease stage, with participants randomly assigned in blocks of four per stratum. Participants, investigators, and sponsor personnel were masked to treatment assignments. Local pharmacists were aware of assignments to support treatment preparation. Pembrolizumab and placebo were administered intravenously once every 3 weeks for up to 17 doses (one before chemoradiotherapy, two during chemoradiotherapy, 14 as maintenance therapy). Chemoradiotherapy included cisplatin (100 mg/m2) administered intravenously once every 3 weeks for two or three doses and accelerated or standard fractionation radiotherapy (70 Gy delivered in 35 fractions). The primary endpoint was event-free survival analysed in all randomly assigned participants. Safety was analysed in all participants who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, NCT03040999, and is active but not recruiting. FINDINGS: Between April 19, 2017, and May 2, 2019, 804 participants were randomly assigned to the pembrolizumab group (n=402) or the placebo group (n=402). 660 (82%) of 804 participants were male, 144 (18%) were female, and 622 (77%) were White. Median study follow-up was 47·7 months (IQR 42·1-52·3). Median event-free survival was not reached (95% CI 44·7 months-not reached) in the pembrolizumab group and 46·6 months (27·5-not reached) in the placebo group (hazard ratio 0·83 [95% CI 0·68-1·03]; log-rank p=0·043 [significance threshold, p≤0·024]). 367 (92%) of 398 participants treated in the pembrolizumab group and 352 (88%) of 398 participants treated in the placebo group had grade 3 or worse adverse events. The most common grade 3 or worse adverse events were decreased neutrophil count (108 [27%] of 398 participants in the pembrolizumab group vs 100 [25%] of 398 participants in the placebo group), stomatitis (80 [20%] vs 69 [17%]), anaemia (80 [20%] vs 61 [15%]), dysphagia (76 [19%] vs 62 [16%]), and decreased lymphocyte count (76 [19%] vs 81 [20%]). Serious adverse events occurred in 245 (62%) participants in the pembrolizumab group versus 197 (49%) participants in the placebo group, most commonly pneumonia (43 [11%] vs 25 [6%]), acute kidney injury (33 [8%] vs 30 [8%]), and febrile neutropenia (24 [6%] vs seven [2%]). Treatment-related adverse events led to death in four (1%) participants in the pembrolizumab group (one participant each from aspiration pneumonia, end-stage renal disease, pneumonia, and sclerosing cholangitis) and six (2%) participants in the placebo group (three participants from pharyngeal haemorrhage and one participant each from mouth haemorrhage, post-procedural haemorrhage, and sepsis). INTERPRETATION: Pembrolizumab plus chemoradiotherapy did not significantly improve event-free survival compared with chemoradiotherapy alone in a molecularly unselected, locally advanced HNSCC population. No new safety signals were seen. Locally advanced HNSCC remains a challenging disease that requires better treatment approaches. FUNDING: Merck Sharp & Dohme, a subsidiary of Merck & Co, Rahway, NJ, USA.

[177Lu]Lu-PSMA-617 plus enzalutamide in patients with metastatic castration-resistant prostate cancer (ENZA-p): an open-label, multicentre, randomised, phase 2 trial.

BACKGROUND: Enzalutamide and lutetium-177 [177Lu]Lu-prostate-specific membrane antigen (PSMA)-617 both improve overall survival in patients with metastatic castration-resistant prostate cancer. Androgen and PSMA receptors have a close intracellular relationship, with data suggesting complementary benefit if targeted concurrently. In this study, we assessed the activity and safety of enzalutamide plus adaptive-dosed [177Lu]Lu-PSMA-617 versus enzalutamide alone as first-line treatment for metastatic castration-resistant prostate cancer. METHODS: ENZA-p was an open-label, randomised, controlled phase 2 trial done at 15 hospitals in Australia. Participants were men aged 18 years or older with metastatic castration-resistant prostate cancer not previously treated with docetaxel or androgen receptor pathway inhibitors for metastatic castration-resistant prostate cancer, gallium-68 [68Ga]Ga-PSMA-PET-CT (PSMA-PET-CT) positive disease, Eastern Cooperative Oncology Group performance status of 0-2, and at least two risk factors for early progression on enzalutamide. Participants were randomly assigned (1:1) by a centralised, web-based system using minimisation with a random component to stratify for study site, disease burden, use of early docetaxel, and previous treatment with abiraterone acetate. Patients were either given oral enzalutamide 160 mg daily alone or with adaptive-dosed (two or four doses) intravenous 7·5 GBq [177Lu]Lu-PSMA-617 every 6-8 weeks dependent on an interim PSMA-PET-CT (week 12). The primary endpoint was prostate-specific antigen (PSA) progression-free survival, defined as the interval from the date of randomisation to the date of first evidence of PSA progression, commencement of non-protocol anticancer therapy, or death. The analysis was done in the intention-to-treat population, using stratified Cox proportional hazards regression. This trial is registered with ClinicalTrials.gov, NCT04419402, and participant follow-up is ongoing. FINDINGS: 162 participants were randomly assigned between Aug 17, 2020, and July 26, 2022. 83 men were assigned to the enzalutamide plus [177Lu]Lu-PSMA-617 group, and 79 were assigned to the enzalutamide group. Median follow-up in this interim analysis was 20 months (IQR 18-21), with 32 (39%) of 83 patients in the enzalutamide plus [177Lu]Lu-PSMA-617 group and 16 (20%) of 79 patients in the enzalutamide group remaining on treatment at the data cutoff date. Median age was 71 years (IQR 64-76). Median PSA progression-free survival was 13·0 months (95% CI 11·0-17·0) in the enzalutamide plus [177Lu]Lu-PSMA-617 group and 7·8 months (95% CI 4·3-11·0) in the enzalutamide group (hazard ratio 0·43, 95% CI 0·29-0·63, p<0·0001). The most common adverse events (all grades) were fatigue (61 [75%] of 81 patients), nausea (38 [47%]), and dry mouth (32 [40%]) in the enzalutamide plus [177Lu]Lu-PSMA-617 group and fatigue (55 [70%] of 79), nausea (21 [27%]), and constipation (18 [23%]) in the enzalutamide group. Grade 3-5 adverse events occurred in 32 (40%) of 81 patients in the enzalutamide plus [177Lu]Lu-PSMA-617 group and 32 (41%) of 79 patients in the enzalutamide group. Grade 3 events that occurred only in the enzalutamide plus [177Lu]Lu-PSMA-617 group included anaemia (three [4%] of 81 participants) and decreased platelet count (one [1%] participant). No grade 4 or 5 events were attributed to treatment on central review in either group. INTERPRETATION: The addition of [177Lu]Lu-PSMA-617 to enzalutamide improved PSA progression-free survival providing evidence of enhanced anticancer activity in patients with metastatic castration-resistant prostate cancer with risk factors for early progression on enzalutamide and warrants further evaluation of the combination more broadly in metastatic prostate cancer. FUNDING: Prostate Cancer Research Alliance (Movember and Australian Federal Government), St Vincent's Clinic Foundation, GenesisCare, Roy Morgan Research, and Endocyte (a Novartis company).

Capivasertib and fulvestrant for patients with hormone receptor-positive, HER2-negative advanced breast cancer (CAPItello-291): patient-reported outcomes from a phase 3, randomised, double-blind, placebo-controlled trial.

BACKGROUND: CAPItello-291 is an ongoing phase 3 trial in which capivasertib-fulvestrant significantly improved progression-free survival versus placebo-fulvestrant in patients with hormone receptor-positive, HER2-negative advanced breast cancer who had relapse or disease progression during or after aromatase inhibitor treatment, in both the overall population and in patients with PIK3CA, AKT1, or PTEN-altered tumours. This study further explored patient-reported health-related quality of life (HRQOL), functioning, symptoms, and symptom tolerability in CAPItello-291. METHODS: This phase 3, randomised, double-blind, placebo-controlled trial, which was conducted across 193 hospitals and cancer centres in 19 countries, enrolled women with any menopausal status or men, aged ≥18 years (≥20 years in Japan), with hormone receptor-positive, HER2-negative locally advanced or metastatic breast cancer who had relapse or disease progression during or after treatment with an aromatase inhibitor, with or without previous cyclin-dependent kinase (CDK) 4 or 6 inhibitor therapy. Patients had an Eastern Cooperative Oncology Group/WHO performance score of 0 or 1 and could have received up to two previous lines of endocrine therapy and up to one previous line of chemotherapy for advanced disease. Patients were randomly assigned (1:1) using block randomisation (stratified according to the presence or absence of liver metastases, previous use of a CDK4/6 inhibitor [yes vs no], and geographical region) to receive oral capivasertib 400 mg (twice daily for 4 days, followed by 3 days off) plus intramuscular fulvestrant 500 mg (every 14 days for the first three injections, then every 28 days) or placebo with matching fulvestrant dosing. The dual primary endpoint of the trial was investigator-assessed progression-free survival assessed both in the overall population and among patients with PIK3CA, AKT1, or PTEN-altered tumours. The EORTC Quality of Life Questionnaire 30-item core module (QLQ-C30) and breast module (QLQ-BR23), Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE), and Patient Global Impression of Treatment Tolerability (PGI-TT) questionnaires were used to assess patient-reported outcomes. Evaluation of EORTC QLQ-C30 and EORTC QLQ-BR23 were secondary endpoints and evaluation of PRO-CTCAE and PGI-TT were pre-defined exploratory endpoints, and these endpoints are the subject of analysis in this Article. Data were collected at baseline and prespecified timepoints. Patient-reported outcomes were analysed in all randomly assigned patients with an evaluable baseline assessment and at least one evaluable post-baseline assessment. Change from baseline was assessed using mixed model with repeated measures for EORTC QLQ-C30 and summarised for QLQ-BR23. Time to deterioration was described using the Kaplan-Meier method. PGI-TT and PRO-CTCAE responses were summarised at each treatment cycle. Patient-reported outcomes were not prospectively powered for statistical comparison. The trial is registered with ClinicalTrials.gov, NCT04305496. FINDINGS: Between June 2, 2020, and Oct 13, 2021, 901 patients were enrolled, of whom 708 patients were randomly assigned to receive capivasertib-fulvestrant (n=355) or placebo-fulvestrant (n=353). The median age of the patients was 59 years (IQR 51-67) in the capivasertib-fulvestrant group and 58 years (IQR 49-66) in the placebo-fulvestrant group. At data cutoff (Aug 15, 2022), the median duration of follow-up for progression-free survival in censored patients was 13·0 months (IQR 9·1-16·7) for capivasertib-fulvestrant and 12·7 months (IQR 2·0-16·4) for placebo-fulvestrant in the overall population. EORTC QLQ-C30 global health status/quality of life (GHS/QOL) scores were maintained from baseline and were similar between treatment groups throughout the study period (difference in mean change from baseline of -2·5 [95% CI -4·5 to -0·6] with capivasertib-fulvestrant vs -5·6 [-7·9 to -3·4] with placebo-fulvestrant; treatment difference 3·1 [95% CI 0·2 to 6·0]). Median time to deterioration in EORTC QLQ-C30 GHS/QOL was 24·9 months (95% CI 13·8 to not reached) in the capivasertib-fulvestrant group and 12·0 months (10·2 to 15·7) in the placebo-fulvestrant group (hazard ratio [HR] 0·70, 95% CI 0·53 to 0·92). Time to deterioration HRs for all EORTC QLQ-C30 and QLQ-BR23 subscale scores showed little difference between the treatment groups, except for diarrhoea, which was worse in the capivasertib-fulvestrant group than in the placebo-fulvestrant group (HR 2·75, 95% CI 2·01-3·81). In PRO-CTCAE symptom assessment, the proportion of patients reporting loose and watery stools "frequently" or "almost constantly" was 29% higher at cycle 1, day 15 in the capivasertib-fulvestrant group than in the placebo-fulvestrant group, decreasing at subsequent cycles. Other PRO-CTCAE-reported symptoms (rash, mouth or throat sores, itchy skin, and numbness or tingling in hands or feet) were absent or mild in most patients in both groups throughout treatment. According to the PGI-TT, most patients in both groups reported "not at all" or "a little bit" of bother from treatment side-effects. INTERPRETATION: Patient-reported outcomes from CAPItello-291 demonstrated that capivasertib-fulvestrant delayed time to deterioration of GHS/QOL and maintained other dimensions of HRQOL (except symptoms of diarrhoea) similarly to fulvestrant. With the clinical efficacy and manageable safety profile, these exploratory results further support the positive benefit-risk profile of capivasertib-fulvestrant in this population. FUNDING: AstraZeneca.

Mapping Conformational Changes in the Saliva Proteome Potentially Associated with Oral Cancer Aggressiveness.

Diverse proteomics-based strategies have been applied to saliva to quantitatively identify diagnostic and prognostic targets for oral cancer. Considering that these targets may be regulated by events that do not imply variation in protein abundance levels, we hypothesized that changes in protein conformation can be associated with diagnosis and prognosis, revealing biological processes and novel targets of clinical relevance. For this, we employed limited proteolysis-mass spectrometry in saliva samples to explore structural alterations, comparing the proteome of healthy control and oral squamous cell carcinoma (OSCC) patients with and without lymph node metastasis. Thirty-six proteins with potential structural rearrangements were associated with clinical patient features including transketolase and its interacting partners. Moreover, N-glycosylated peptides contribute to structural rearrangements of potential diagnostic and prognostic markers. Altogether, this approach utilizes saliva proteins to search for targets for diagnosing and prognosing oral cancer and can guide the discovery of potential regulated sites beyond protein-level abundance.