Ultrasensitive plasma-based monitoring of tumor burden using machine-learning-guided signal enrichment.

In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole-genome sequencing (WGS). Here we now introduce MRD-EDGE, a machine-learning-guided WGS ctDNA single-nucleotide variant (SNV) and copy-number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by ~300× compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, vastly expanding its applicability within solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in TF in response to neoadjuvant immunotherapy in lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables plasma-only (non-tumor-informed) disease monitoring in advanced melanoma and lung cancer, yielding clinically informative TF monitoring for patients on immune-checkpoint inhibition.

Genotype-to-phenotype mapping of somatic clonal mosaicism via single-cell co-capture of DNA mutations and mRNA transcripts.

Somatic mosaicism is a hallmark of malignancy that is also pervasively observed in human physiological aging, with clonal expansions of cells harboring mutations in recurrently mutated driver genes. Bulk sequencing of tissue microdissection captures mutation frequencies, but cannot distinguish which mutations co-occur in the same clones to reconstruct clonal architectures, nor phenotypically profile clonal populations to delineate how driver mutations impact cellular behavior. To address these challenges, we developed single-cell Genotype-to-Phenotype sequencing (scG2P) for high-throughput, highly-multiplexed, single-cell joint capture of recurrently mutated genomic regions and mRNA phenotypic markers in cells or nuclei isolated from solid tissues. We applied scG2P to aged esophagus samples from five individuals with high alcohol and tobacco exposure and observed a clonal landscape dominated by a large number of clones with a single driver event, but only rare clones with two driver mutations. NOTCH1 mutants dominate the clonal landscape and are linked to stunted epithelial differentiation, while TP53 mutants and double-driver mutants promote clonal expansion through both differentiation biases and increased cell cycling. Thus, joint single-cell highly multiplexed capture of somatic mutations and mRNA transcripts enables high resolution reconstruction of clonal architecture and associated phenotypes in solid tissue somatic mosaicism.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization.

Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80highSca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy.

Integration of whole transcriptome spatial profiling with protein markers.

Spatial transcriptomics and proteomics provide complementary information that independently transformed our understanding of complex biological processes. However, experimental integration of these modalities is limited. To overcome this, we developed Spatial PrOtein and Transcriptome Sequencing (SPOTS) for high-throughput simultaneous spatial transcriptomics and protein profiling. Compared with unimodal measurements, SPOTS substantially improves signal resolution and cell clustering and enhances the discovery power in differential gene expression analysis across tissue regions.

Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation.

Somatic mutations in cancer genes have been detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, because mutated and wild-type cells are admixed, we have limited ability to link genotypes with phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing genotype, transcriptomes and methylomes in progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations result in myeloid over lymphoid bias, and an expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate, with dysregulated expression of lineage and leukemia stem cell markers. Mutated DNMT3A leads to preferential hypomethylation of polycomb repressive complex 2 targets and a specific CpG flanking motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics paves the road to defining the downstream consequences of mutations that drive clonal mosaicism.

[Prescription appropriateness in elderly patients with polypharmacy in primary care: Cluster-randomized controlled trial PHARM-PC]. / Adecuación de la prescripción en pacientes mayores polimedicados en atención primaria. Ensayo clínico controlado aleatorizado por grupos PHARM-PC.

OBJECTIVES: To assess the effectiveness of a pharmacist-led systematic review of medications on: potentially inappropriate medications (PIM), health outcomes and costs. DESIGN: Prospective, open, controlled and cluster-randomized clinical trial. SETTING: Six primary care clinics from Balearic Islands. PARTICIPANTS: Forty-two clusters (21 per group), and 549 patients aged ≥65 years and ≥5 chronic medications were included; of which 277 were allocated to Intervention Group (IG) and 272 to Control Group (CG). Patients were excluded if they were: institutionalized, temporarily displaced, routinely monitored under private healthcare, or home care. INTERVENTION: PIM detection by the pharmacist using a combination of explicit and implicit methods; and communication of the most appropriate therapeutic strategies to the physician. MEASUREMENTS: Proportion of patients with PIM and mean number of PIM/patient (main outcomes); and morbidity, mortality, and costs (secondary outcomes) were assessed. STATISTICAL PLAN: Following an intention-to-treat approach, quantitative and qualitative outcomes variables were compared by T-Student and Chi-square tests, respectively. Results were providing as difference in proportions for qualitative outcomes and difference in means for quantitative outcomes with respective 95% confidence intervals (95% CI). RESULTS: After intervention, proportion of patients with PIM decreased by 13.7% (95% CI: 9.3; 18.2) more in IG than CG. Mean number of PIM/patient and mean cost of PIM/patient decreased by 0.43 (95% CI: 0.32; 0.54) and 72.11€ (95% CI: 26.15; 118.06) more in IG than CG, respectively. However, no statistically significant differences were observed in morbidity, mortality or costs of healthcare resources. CONCLUSIONS: PIM detection and recommendations provided by pharmacist could contribute to reduce significantly PIM and drug expenditure; but without reaching statistically significant differences in morbidity, mortality, and healthcare resources costs.

Pharmacist-led intervention on potentially inappropriate prescription in patients with polypharmacy: PHARM-PC clinical trial protocol.

OBJECTIVE: Polypharmacy and potentially inappropriate medications (that  is, those associated with an unfavorable risk-benefit ratio) are common concerns in the context of elderly patients treated in primary care as they may increase the risk of morbidity and mortality, as well as  healthcare costs. Several studies have assessed the impact of pharmacist- led systematic reviews with respect to prescription appropriateness, health outcomes and/or costs. However, no cluster-randomized controlled  trial has been identified that provides an overall assessment of these  variables. The objective is to determine the effectiveness of a pharmacist- led systematic medication review in reducing the mean number and proportion of patients on potentially inappropriate medications (primary  goal); as well as in decreasing morbidity and mortality and the cost of  medications and the use of healthcare resources (secondary goals). METHOD: An open-label, cluster-randomized controlled trial will be conducted; where primary care physicians will be randomized either to receive (intervention group) or not to receive pharmacist  recommendations to withdraw potentially inappropriate medications  detected through the combined use of explicit and implicit criteria (control  group). Primary end-points will be the proportion of patients on potentially inappropriate medications and the mean number of such medications per  patient. Secondary endpoints will be healthcare resources used, the  proportion of patients who die and the mean number of days survived, as  well as the cost of medications and cost of healthcare resources used. CONCLUSIONS: In line with similar reports and based on our study´s design, we hope to obtain statistically significant reductions in the use  of potentially inappropriate medications and in medication costs overall.  We do not however expect to obtain significant reductions in  morbimortality or the cost of health resources employed.

Objetivo: La polimedicación y la medicación potencialmente inapropiada (que presenta balance beneficio-riesgo desfavorable) son  importantes preocupaciones respecto a los pacientes mayores en atención  primaria, ya que pueden incrementar el riesgo de morbimortalidad y los  costes sanitarios. Diversos estudios han evaluado el impacto de la revisión  sistemática de la medicación conducida por el farmacéutico sobre variables de adecuación, recursos sanitarios y/o costes. Sin embargo, no se han  encontrado ensayos controlados aleatorizados por clúster que evalúen  globalmente todas estas variables. El objetivo de este estudio es  determinar el impacto de una revisión sistemática de medicación  conducida por el farmacéutico para reducir el número medio y la  proporción de pacientes con medicación potencialmente inapropiada  (objetivo principal), así como para reducir la morbimortalidad y los costes  (objetivos secundarios).Método: Se realizará un ensayo clínico abierto, controlado y aleatorizado por clústeres, donde los médicos de atención primaria, en  representación de sus respectivos cupos de pacientes, serán aleatorizados a recibir recomendaciones del farmacéutico para retirar  medicaciones potencialmente inapropiadas detectadas mediante  combinación de métodos implícitos y explícitos (grupo intervención) o no  recibirlas (grupo control). Las variables primarias serán la proporción de  pacientes y el número medio de medicaciones potencialmente  inapropiadas por paciente. Las variables secundarias serán los recursos  asistenciales frecuentados, proporción de pacientes fallecidos y días de  supervivencia; y costes de medicamentos y de recursos asistenciales.Conclusiones: Análogamente a estudios similares, y en base al diseño de  nuestro estudio, esperamos obtener reducción estadísticamente  ignificativa para medicaciones potencialmente inapropiadas y costes de medicamentos. Sin embargo, no esperamos diferencias significativas en morbimortalidad ni en costes de recursos asistenciales.

Houston hurricane Harvey health (Houston-3H) study: assessment of allergic symptoms and stress after hurricane Harvey flooding.

BACKGROUND: In August 2017, Hurricane Harvey caused unprecedented flooding across the greater Houston area. Given the potential for widespread flood-related exposures, including mold and sewage, and the emotional and mental toll caused by the flooding, we sought to evaluate the short- and long-term impact of flood-related exposures on the health of Houstonians. Our objectives were to assess the association of flood-related exposures with allergic symptoms and stress among Houston-area residents at two time points: within approximately 30 days (T1) and 12 months (T2) after Hurricane Harvey's landfall. METHODS: The Houston Hurricane Harvey Health (Houston-3H) Study enrolled a total of 347 unique participants from four sites across Harris County at two times: within approximately 1-month of Harvey (T1, n = 206) and approximately 12-months after Harvey (T2, n = 266), including 125 individuals who participated at both time points. Using a self-administered questionnaire, participants reported details on demographics, flood-related exposures, and health outcomes, including allergic symptoms and stress. RESULTS: The majority of participants reported hurricane-related flooding in their homes at T1 (79.1%) and T2 (87.2%) and experienced at least one allergic symptom after the hurricane (79.4% at T1 and 68.4% at T2). In general, flood-exposed individuals were at increased risk of upper respiratory tract allergic symptoms, reported at both the T1 and T2 time points, with exposures to dirty water and mold associated with increased risk of multiple allergic symptoms. The mean stress score of study participants at T1 was 8.0 ± 2.1 and at T2, 5.1 ± 3.2, on a 0-10 scale. Participants who experienced specific flood-related exposures reported higher stress scores when compared with their counterparts, especially 1 year after Harvey. Also, a supplementary paired-samples analysis showed that reports of wheezing, shortness of breath, and skin rash did not change between T1 and T2, though other conditions were less commonly reported at T2. CONCLUSION: These initial Houston-3H findings demonstrate that flooding experiences that occurred as a consequence of Hurricane Harvey had lasting impacts on the health of Houstonians up to 1 year after the hurricane.

Utility of a trigger tool (TRIGGER-CHRON) to detect adverse events associated with high-alert medications in patients with multimorbidity.

OBJECTIVE: To determine the utility of a tool (TRIGGER-CHRON) for identifying adverse drug events (ADEs) associated with the administration of high-alert medications in elderly patients with multimorbidity and to determine the medications most frequently implicated. METHODS: A retrospective observational study was conducted at 12 Spanish hospitals. A random sample of five medical records from each hospital was selected weekly for review over a 12-week period. We included patients aged 65 and over with multimorbidities, hospitalised for >48 hours. ADEs detected by the 32 TRIGGER-CHRON signals and caused by high-alert medications included on the Spanish HAMC list for chronic patients were selected for analysis. Triggers identified and ADEs detected were recorded. The severity and preventability of the ADEs were evaluated. The positive predictive value (PPV) of each trigger was calculated. RESULTS: On 720 charts reviewed, 908 positive triggers were identified that led to the detection of 158 ADEs caused by at least one high-alert medication on the HAMC list. These ADEs occurred in 139 patients (prevalence 19.3/100 admissions). The majority of ADEs were mild and 59.5% were deemed preventable. The drugs most frequently associated with ADEs were corticosteroids, loop diuretics, opioid analgesics and oral anticoagulants. Fifteen triggers had PPVs ≥20%. Six triggers (serum glucose >110 mg/dL, abrupt cessation of medication, oversedation/lethargy, hypotension, adverse reaction recorded and constipation) accounted for 69.8% of the ADEs identified. CONCLUSIONS: Applying the TRIGGER-CHRON to hospitalised patients with multimorbidity in 12 Spanish centres allowed detection of one adverse event caused by a high-alert drug for every four patients, which were preventable in a large proportion of patients. This confirms the need to establish interventions that reduce harm with these medications. We believe that TRIGGER-CHRON can be a useful tool to measure this harm and to determine the effects of medication safety improvement programmes as they are implemented.

Focal Plane Shift Imaging for the Analysis of Dynamic Wetting Processes.

Droplet-surface interactions are common to a plethora of natural and industrial processes due to their ability to rapidly exchange energy, mass, and momentum. Droplets are particularly of interest due to their large surface-to-volume ratios and hence enhanced transport properties. For example, coalescence-induced droplet jumping on superhydrophobic surfaces has recently received much attention for its potential to enhance heat transfer, anti-icing, and self-cleaning performance by passively shedding microscale water droplets. To study droplet jumping, researchers typically use a two-camera setup to observe the out-of-plane droplet motion, with limited success due to the inability to resolve the depth dimension using two orthogonal cameras. Here we develop a single-camera technique capable of providing three-dimensional (3D) information through the use of focal plane manipulation, termed "focal plane shift imaging" (FPSI). We used FPSI to study the jumping process on superhydrophobic surfaces having a wide range of structure length scales (10 nm < l < 1 µm) and droplet radii (3 µm < R < 160 µm). We benchmarked the FPSI technique and studied the effects of droplet mismatch, multidroplet coalescence, and multihop coalescence on droplet jumping speed. Furthermore, we were able to resolve the full 3D trajectory of multiple jumping events, to show that, unlike previously theorized, the departure angle during droplet jumping is not a function of droplet mismatch or number of droplets coalescing prior to jumping. Rather, angular deviation arises due to in-plane motion postcoalescence governed by droplet pinning. The outcomes of this work both elucidate key fundamental aspects governing droplet jumping and provide a powerful imaging platform for the study of dynamic droplet processes that result in out-of-plane motion such as sliding, coalescence, or impact.

[Development of integrated support software for clinical nutrition]. / Desarrollo de una aplicación informática de ayuda al soporte nutricional especializado integrado en la historia clínica electrónica.

OBJECTIVES: to develop an integrated computer software application for specialized nutritional support, integrated in the electronic clinical record, which detects automatically and early those undernourished patients or at risk of developing undernourishment, determining points of opportunity for improvement and evaluation of the results. METHODS: the quality standards published by the Nutrition Work Group of the Spanish Society of Hospital Pharmacy (SEFH) and the recommendations by the Pharmacy Group of the Spanish Society of Parenteral and Enteral Nutrition (SENPE) have been taken into account. According to these quality standards, the nutritional support has to include the following healthcare stages or sub-processes: nutritional screening, nutritional assessment, plan for nutritional care, prescription, preparation and administration. RESULTS: this software allows to conduct, in an automated way, a specific nutritional assessment for those patients with nutritional risk, implementing, if necessary, a nutritional treatment plan, conducting follow-up and traceability of outcomes derived from the implementation of improvement actions, and quantifying to what extent our practice is close to the established standard. CONCLUSIONS: this software allows to standardize the specialized nutritional support from a multidisciplinary point of view, introducing the concept of quality control per processes, and including patient as the main customer.

Objetivos: desarrollar una aplicacion informatica integral en el soporte nutricional especializado, e integrado en la historia clinica electronica, que detecte de forma automatizada y precoz a los pacientes desnutridos o en riesgo de desarrollar desnutricion, determinando puntos de oportunidad de mejora y evaluacion de resultados. Métodos: se han tenido en cuenta los estandares de calidad publicados por el grupo de trabajo de nutricion de la Sociedad Espanola de Farmacia Hospitalaria (SEFH) y las recomendaciones del grupo de farmacia de la Sociedad Espanola de Nutricion Parenteral y Enteral (SENPE). De acuerdo con dichos estandares de calidad, las etapas o subprocesos asistenciales que debe contemplar el soporte nutricional son: cribado nutricional, valoracion nutricional, plan de cuidados nutricionales, formulacion, elaboracion y administracion. Resultados: la aplicacion permite, de forma automatizada, realizar una valoracion nutricional especifica a los pacientes con riesgo nutricional, instaurando, si fuese preciso, un plan de tratamiento nutricional y realizando el seguimiento y trazabilidad de los resultados derivados de la implantacion de acciones de mejora y, cuantificando en que medida nuestra practica se aproxima a la establecida como estandar. Conclusiones: la aplicacion permite estandarizar el soporte nutricional especializado desde un punto multidisciplinar, introduciendo el concepto de control de calidad por procesos y al paciente como cliente principal.