Long-term alliance for pathology equipment and technology acquisition: a paradigm shift for improved quality and efficiency.

OBJECTIVE: Personnel costs are the largest single budget item in the clinical laboratory, other major expenses being equipment, analyzers, blood and blood components, and cost of day-to-day consumables. This report describes our experience with developing a long-term relationship with a single major vendor as a paradigm shift from the traditional multiple vendors, multiple contracts, and recurrent extended negotiations. Our objective was to develop a long-term approach for replacement of effete equipment and upgrades to operations in a pathology and laboratory medicine department in collaboration with vendors providing equipment and services. METHODS: Major vendors were invited to visit the department to analyze the workload and workflow and suggest integrated solutions to meet the goals of the department. Multiple iterations of the proposals were evaluated, and a recommendation made to the medical center leadership. The vendor, the medical center, and the department jointly developed a 15-year partnership plan to improve the operations of pathology services. The agreement encompasses a range of management and performance criteria for both sides. The salient items discussed were laboratory staffing, turnaround time, workload change, test insourcing, reference laboratory costs, and scholarly productivity and teaching. RESULTS: The agreement reduced laboratory staffing by 21%, eliminated stat tests by reducing the turnaround time for routine tests to less than 45 minutes for 90% of tests, with an increase of 9.1% in the number of tests, Cost avoidance in salary and reference laboratory costs was $3,424,136/year against an expected target of $2 million in total savings, despite not including cost avoidance from promoting appropriate use of laboratory testing for inpatients and increase in revenue from increase in ambulatory testing. Vizient score in laboratory utilization improved from the 94th to 76th percentile. Scholarly output increased by more than 100%. CONCLUSION: This model of a long-term alliance with a chosen vendor led to improvements in quality and efficiency.

Dynamic Coronary Blood Flow Velocity and Wall Shear Stress Estimation Using Ultrasound in an Ex Vivo Porcine Heart.

PURPOSE: Wall shear stress (WSS) is a critically important physical factor contributing to atherosclerosis. Mapping the spatial distribution of local, oscillatory WSS can identify important mechanisms underlying the progression of coronary artery disease. METHODS: In this study, blood flow velocity and time-varying WSS were estimated in the left anterior descending (LAD) coronary artery of an ex vivo beating porcine heart using ultrasound with an 18 MHz linear array transducer aligned with the LAD in a forward-viewing orientation. A pulsatile heart loop with physiologically-accurate flow was created using a pulsatile pump. The coronary artery wall motion was compensated using a local block matching technique. Next, 2D and 3D velocity magnitude and WSS maps in the LAD coronary artery were estimated at different time points in the cardiac cycle using an ultrafast Doppler approach. The blood flow velocity estimated using the presented approach was compared with a commercially-available, calibrated single element blood flow velocity measurement system. RESULTS: The resulting root mean square error (RMSE) of 2D velocity magnitude acquired from a high frequency, linear array transducer was less than 8% of the maximum velocity estimated by the commercial system. CONCLUSION: When implemented in a forward-viewing intravascular ultrasound device, the presented approach will enable dynamic estimation of WSS, an indicator of plaque vulnerability in coronary arteries.

ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.

Transapical ventricular reshaping reduces functional mitral regurgitation and improves ventricular function in a preclinical model of ischemic cardiomyopathy.

OBJECTIVE: A significant proportion of patients with advanced heart failure present with dilated left ventricles and functional mitral regurgitation. These patients currently have limited treatment options. The MitraClip device (Abbott) has benefited only patients with smaller left ventricles (end-diastolic dimension <70 mm), whereas those with larger left ventricles did not benefit. A possible explanation is correcting functional mitral regurgitation alone may not adequately reduce the wall stresses of a dilated left ventricle. We have developed a beating-heart device that not only approximates the papillary muscles to reduce functional mitral regurgitation but also modifies the left ventricle size and shape to reduce wall stress. METHODS: Yorkshire swine (n = 16) had a myocardial infarction induced by permanent occlusion of the left circumflex with intracoronary ethanol. Three months later, the animals developed heart failure and moderate or greater functional mitral regurgitation. Through a transapical approach, the new device was implanted under echocardiography guidance to reshape the left ventricle and correct functional mitral regurgitation. The acute impact of this approach on the mitral valve and left ventricle was assessed with echocardiography and invasive hemodynamics. RESULTS: After reshaping, echocardiography showed a decrease in end-diastolic volume by 36.3 ± 30.5 mL (P < .001), a decrease in sphericity index by 0.143 ± 0.087 (P < .001), and an increase in ejection fraction of 5.90% ± 6.38% (P < .01). Mitral valve tenting area was reduced by 39.29 ± 33.66 mm2 (P < .001), coaptation length was increased by 2.12 ± 1.02 mm (P < .001), and posterior excursion angle was improved by 9.07° ± 9.14° (P < .01), resulting in functional mitral regurgitation reduction. CONCLUSIONS: Correction of functional mitral regurgitation with favorable changes in mitral valve geometry and reduction in left ventricle geometry is possible with the proposed device.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/ßc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/ßc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/ßc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance. SIGNIFICANCE: Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.

Ultra-High-Molecular-Weight Poly(Dioxolane): Enhancing the Mechanical Performance of a Chemically Recyclable Polymer.

We report a method to synthesize ultra-high-molecular-weight poly(1,3-dioxolane) (UHMW pDXL), a chemically recyclable thermoplastic material with excellent physical properties. We aimed to enhance the mechanical properties of sustainable polymers by increasing the molecular weight and found that UHMW pDXL exhibits similar tensile properties to ultra-high-molecular-weight polyethylene (UHMWPE). The new polymerization method uses metal-free and economically friendly initiators to achieve UHMW pDXL with molecular weights greater than 1000 kDa. The development of UHMW pDXL provides a potential solution for capturing value from plastic waste and addressing the detrimental effects of plastic waste.

Full-field strain mapping of healthy and pathological mouse aortas using stereo digital image correlation.

The murine aorta is a complex, heterogeneous structure that undergoes large and sometimes asymmetrical deformations under loading. For analytical convenience, mechanical behavior is predominantly described using global quantities that fail to capture critical local information essential to elucidating aortopathic processes. Here, in our methodological study, we used stereo digital image correlation (StereoDIC) to measure the strain profiles of speckle-patterned healthy and elastase-infused, pathological mouse aortas submerged in a temperature-controlled liquid medium. Our unique device rotates two 15-degree stereo-angle cameras that gather sequential digital images while simultaneously performing conventional biaxial pressure-diameter and force-length testing. A StereoDIC Variable Ray Origin (VRO) camera system model is employed to correct for high-magnification image refraction through hydrating physiological media. The resultant Green-Lagrange surface strain tensor was quantified at different blood vessel inflation pressures, axial extension ratios, and after aneurysm-initiating elastase exposure. Quantified results capture large, heterogeneous, inflation-related, circumferential strains that are drastically reduced in elastase-infused tissues. Shear strains, however, were very small on the tissue's surface. Spatially averaged StereoDIC-based strains were generally more detailed than those determined using conventional edge detection techniques.

Sustainable financing for Immunization Agenda 2030.

Sustainable financing for immunization refers to the sufficient and predictable allocation and use of resources to support the achievement of immunization goals within the framework of overall health financing. The Immunization Agenda 2030 (IA2030) agenda spells out four important focus areas needed for sustainable financing: (1) ensuring sufficient and predictable resources, (2) making optimal use of resources, (3) aligning partnerships, and (4) supporting sustainable transitions from external assistance. This paper summarizes the evidence and proposes interventions under each area. While immunization is one of the best investments and justifies public financing, the COVID-19 pandemic has led to the worst economic recession since the Great Depression and threatens countries' ability to mobilize funding to ensure continuity and access to essential services, including immunization. Strategies for ensuring adequate resources differ by income group but include raising more revenues, reprioritizing the budget towards health, and ensuring that health resources favor Primary Health Care (PHC) and immunization. In low- and lower-middle income countries, support from Gavi, the Vaccine Alliance, which channels the largest amount of external financing, will remain important, but some lower-middle income countries will need to prepare for transition. Countries benefitting from the Global Polio Eradication Initiative (GPEI) are also experiencing a transition from GPEI financing to domestic and other external financing. This paper outlines ways in which countries can improve the use of domestic and external resources to better incentivize high-quality PHC and immunization services and align immunization programs with health sector reforms. While governments must lead, collective action from development partners, the private sector, and civil society is needed to promote health system financing systems that ensure that the world is better prepared for future outbreaks and pandemics, while reinforcing the IA2030 vision and making progress towards universal health coverage and the Sustainable Development Goals.

Breast cancer survivors with preserved or rescued cardiorespiratory fitness have similar cardiac, pulmonary and muscle function compared to controls.

Breast cancer survivors (BCS) have a high prevalence of cardiovascular disease and low cardiorespiratory fitness (CRF). CRF is an important predictor of survival in BCS. However, the physiological factors that contribute to low CRF in BCS have not been completely elucidated. To assess differences in physiological factors (cardiac, pulmonary, muscle function) related to CRF between BCS and controls. Twenty-three BCS and 23 age-body mass index (BMI) matched controls underwent a peak cycling exercise test to determine CRF, with physiological factors measured at resting and at peak exercise. Cardiac hemodynamics (stroke volume [SV], SVindex, heart rate [HR], cardiac output [Formula: see text], and [Formula: see text]index) were evaluated using ultrasonography. Pulmonary function was evaluated using the oxygen uptake efficiency slope (OUES), ventilation to carbon dioxide production slope [Formula: see text] and breathing reserve at peak exercise (BR). Muscle oxygenation variables (oxygenated [HbO2] deoxygenated [HHb] and total hemoglobin [Hb], and tissue oxygenation index [TSI]) were measured with near-infrared spectroscopy (NIRS). Both groups had similar CRF and similarly increased all hemodynamic variables (HR, SV, SVindex, [Formula: see text] and [Formula: see text]index) at peak exercise compared to resting (p < 0.001). BCS had higher overall HR and lower SVindex (group effect, p < 0.05). BCS had similar OUES, [Formula: see text] and BR compared to the controls. Both groups decreased TSI, and increased Hb and HHb similarly at peak exercise compared to resting (p < 0.001). Our data suggest BCS do not exhibit differences in cardiac, pulmonary, or muscle function at peak exercise compared to controls, when both groups have similar CRF and physical activity.

Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia.

The interaction of germline variation and somatic cancer driver mutations is under-investigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondrially-encoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 (IDH1), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1-mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.

A Biohybrid Material With Extracellular Matrix Core and Polymeric Coating as a Cell Honing Cardiovascular Tissue Substitute.

Objective: To investigate the feasibility of a hybrid material in which decellularized pericardial extracellular matrix is functionalized with polymeric nanofibers, for use as a cardiovascular tissue substitute. Background: A cardiovascular tissue substitute, which is gradually resorbed and is replaced by host's native tissue, has several advantages. Especially in children and young adults, a resorbable material can be useful in accommodating growth, but also enable rapid endothelialization that is necessary to avoid thrombotic complications. In this study, we report a hybrid material, wherein decellularized pericardial matrix is functionalized with a layer of polymeric nanofibers, to achieve the mechanical strength for implantation in the cardiovascular system, but also have enhanced cell honing capacity. Methods: Pericardial sacs were decellularized with sodium deoxycholate, and polycaprolactone-chitosan fibers were electrospun onto the matrix. Tissue-polymer interaction was evaluated using spectroscopic methods, and the mechanical properties of the individual components and the hybrid material were quantified. In-vitro blood flow loop studies were conducted to assess hemocompatibility and cell culture methods were used to assess biocompatibility. Results: Encapsulation of the decellularized matrix with 70 µm thick matrix of polycaprolactone-chitosan nanofibers, was feasible and reproducible. Spectroscopy of the cross-section depicted new amide bond formation and C-O-C stretch at the interface. An average peel strength of 56.13 ± 11.87 mN/mm2 was measured, that is sufficient to withstand a high shear of 15 dynes/cm2 without delamination. Mechanical strength and extensibility ratio of the decellularized matrix alone were 18,000 ± 4,200 KPa and 0.18 ± 0.03% whereas that of the hybrid was higher at 20,000 ± 6,600 KPa and 0.35 ± 0.20%. Anisotropy index and stiffness of the biohybrid were increased as well. Neither thrombus formation, nor platelet adhesion or hemolysis was measured in the in-vitro blood flow loop studies. Cellular adhesion and survival were adequate in the material. Conclusion: Encapsulating a decellularized matrix with a polymeric nanofiber coating, has favorable attributes for use as a cardiovascular tissue substitute.

Better Understanding of the Metamorphosis of Pregnancy (BUMP): protocol for a digital feasibility study in women from preconception to postpartum.

The Better Understanding the Metamorphosis of Pregnancy (BUMP) study is a longitudinal feasibility study aimed to gain a deeper understanding of the pre-pregnancy and pregnancy symptom experience using digital tools. The present paper describes the protocol for the BUMP study. Over 1000 participants are being recruited through a patient provider-platform and through other channels in the United States (US). Participants in a preconception cohort (BUMP-C) are followed for 6 months, or until conception, while participants in a pregnancy cohort (BUMP) are followed into their fourth trimester. Participants are provided with a smart ring, a smartwatch (BUMP only), and a smart scale (BUMP only) alongside cohort-specific study apps. Participant centric engagement strategies are used that aim to co-design the digital approach with participants while providing knowledge and support. The BUMP study is intended to lay the foundational work for a larger study to determine whether participant co-designed digital tools can be used to detect, track and return multimodal symptoms during the perinatal window to inform individual level symptom trajectories.

Alcohol Consumption: A New Risk Factor for Arterial Stiffness?

The relationship between alcohol consumption and cardiovascular disease risk is complex. Low-to-moderate daily alcohol consumption (1-2 drinks/day) is associated with reduced risk, whereas greater amounts of alcohol consumption and a "binge" pattern of drinking are associated with increased cardiovascular risk and mortality. Arterial stiffness may help explain the complex relationship. This integrated review summarizes data from studies examining the associations between alcohol consumption and pulse wave velocity, a gold standard measure of arterial stiffness. We also briefly review the concept and methodology of pulse wave velocity measurement as well as the mechanisms of alcohol-induced arterial stiffening. Findings among the different studies reviewed were inconsistent with methodological challenges related to alcohol use assessment. While making specific conclusions regarding this relationship is tenuous; the data suggest that excessive alcohol consumption or a binge drinking pattern is associated with increased arterial stiffness.

The Effects of a Pain Management-Focused Mobile Health Behavior Intervention on Older Adults' Self-efficacy, Satisfaction with Functioning, and Quality of Life: a Randomized Pilot Trial.

BACKGROUND: This report investigates the impact of a remote physical activity intervention on self-efficacy, satisfaction with functioning, and health-related quality of life (HRQOL) as assessed by the SF-36 in obese older adults with chronic pain. The intervention was group-mediated in nature and based in social cognitive theory and mindfulness-based relapse prevention. METHODS: Participants (N = 28; 70.21 ± 5.22 years) were randomly assigned to receive either the active intervention, which focused on reducing caloric intake and increasing steps across the day or to a waitlist control condition. RESULTS: Over 12 weeks, intervention participants reported a moderate, positive improvement in self-efficacy for walking relative to control. They also reported large magnitude improvements in satisfaction for physical functioning as well as improvements on pain and the physical functioning subscales of the SF-36. CONCLUSIONS: These findings expand on previous research showing similar effects in response to structured exercise, this time via a protocol that is likely to be scalable and sustainable for many older adults. Additional work on larger and more diverse samples is warranted.

Effect of live-fire training on ventricular-vascular coupling.

INTRODUCTION: Cardiovascular events are a leading cause of firefighter duty-related death, with the greatest risk occurring during or shortly after fire suppression activity. Increased cardiovascular risk potentially manifests from detrimental changes in ventricular function, vascular load, and their interaction, described as ventricular-vascular coupling. PURPOSE: To determine the effect of live-fire training on ventricular-vascular coupling. METHODS: Sixty-eight male (28 [Formula: see text] 7 years, 26.9 [Formula: see text] 3.9 kg/m2) and fifteen female (36 [Formula: see text] 8 years, 24.3 [Formula: see text] 3.9 kg/m2) firefighters completed hemodynamic and cardiac measures before and after 3 h of intermittent live-fire training. Left ventricular function was assessed as ejection fraction (EF) and ventricular elastance (ELV: end systolic pressure [ESP]/end systolic volume) via echocardiography and applanation tonometry-estimated ESP. Vascular load was assessed as arterial elastance (EA: ESP/stroke volume [SV]). Ventricular-vascular coupling (VVC) was quantified as the ratio of EA to ELV and indexed to body surface area (EAI, ELVI). RESULTS: Following firefighting EF decreased (p < 0.01) with no change in ELVI (p = 0.34). SV decreased (p < 0.01) with no change in ESP (p = 0.09), driving a significant increase in EAI (p < 0.01). These changes resulted in a significant increase in the VVC ratio (p < 0.01). CONCLUSION: The findings suggest that firefighting does not alter ventricular elastance but increases arterial elastance in healthy firefighters, resulting in a mismatch between ventricular and vascular systems. This increase in ventricular-vascular coupling ratio and concomitant reduction in ventricular systolic function may contribute to increased cardiovascular risk following live firefighting.

The oral microbiome in alcohol use disorder: a longitudinal analysis during inpatient treatment.

BACKGROUND: Alcohol use disorder (AUD)-induced disruption of oral microbiota can lead to poor oral health; there have been no studies published examining the longitudinal effects of alcohol use cessation on the oral microbiome. AIM: To investigate the oral microbiome during alcohol cessation during inpatient treatment for AUD. METHODS: Up to 10 oral tongue brushings were collected from 22 AUD patients during inpatient treatment at the National Institutes of Health. Alcohol use history, smoking, and periodontal disease status were measured. Oral microbiome samples were sequenced using 16S rRNA gene sequencing. RESULTS: Alpha diversity decreased linearly during treatment across the entire cohort (P = 0.002). Alcohol preference was associated with changes in both alpha and beta diversity measures. Characteristic tongue dorsum genera from the Human Microbiome Project such as Streptococcus, Prevotella, Veillonella and Haemophilus were highly correlated in AUD. Oral health-associated genera that changed longitudinally during abstinence included Actinomyces, Capnocytophaga, Fusobacterium, Neisseria and Prevotella. CONCLUSION: The oral microbiome in AUD is affected by alcohol preference. Patients with AUD often have poor oral health but abstinence and attention to oral care improve dysbiosis, decreasing microbiome diversity and periodontal disease-associated genera while improving acute oral health.

Commensal gut bacterium critically regulates alveolar bone homeostasis.

The alveolar bone is a unique osseous tissue due to the presence of the teeth and the proximity of commensal oral microbes. Commensal microbe effects on alveolar bone homeostasis have been attributed to the oral microbiota, yet the impact of commensal gut microbes is unknown. Study purpose was to elucidate whether commensal gut microbes regulate osteoimmune mechanisms and skeletal homeostasis in alveolar bone. Male C57BL/6T germfree (GF) littermate mice were maintained as GF or monoassociated with segmented filamentous bacteria (SFB), a commensal gut bacterium. SFB has been shown to elicit broad immune response effects, including the induction of TH17/IL17A immunity, which impacts the development and homeostasis of host tissues. SFB colonized the gut, but not oral cavity, and increased IL17A levels in the ileum and serum. SFB had catabolic effects on alveolar bone and non-oral skeletal sites, which was attributed to enhanced osteoclastogenesis. The alveolar bone marrow of SFB vs. GF mice had increased dendritic cells, activated helper T-cells, TH1 cells, TH17 cells, and upregulated Tnf. Primary osteoblast cultures from SFB and GF mice were stimulated with vehicle-control, IL17A, or TNF to elucidate osteoblast-derived signaling factors contributing to the pro-osteoclastic phenotype in SFB mice. Treatment of RAW264.7 osteoclastic cells with supernatants from vehicle-stimulated SFB vs. GF osteoblasts recapitulated the osteoclast phenotype found in vivo. Supernatants from TNF-stimulated osteoblasts normalized RAW264.7 osteoclast endpoints across SFB and GF cultures, which was dependent on the induction of CXCL1 and CCL2. This report reveals that commensal gut microbes have the capacity to regulate osteoimmune processes in alveolar bone. Outcomes from this investigation challenge the current paradigm that alveolar bone health and homeostasis is strictly regulated by oral microbes.

Clonal architecture predicts clinical outcomes and drug sensitivity in acute myeloid leukemia.

The impact of clonal heterogeneity on disease behavior or drug response in acute myeloid leukemia remains poorly understood. Using a cohort of 2,829 patients, we identify features of clonality associated with clinical features and drug sensitivities. High variant allele frequency for 7 mutations (including NRAS and TET2) associate with dismal prognosis; elevated GATA2 variant allele frequency correlates with better outcomes. Clinical features such as white blood cell count and blast percentage correlate with the subclonal abundance of mutations such as TP53 and IDH1. Furthermore, patients with cohesin mutations occurring before NPM1, or transcription factor mutations occurring before splicing factor mutations, show shorter survival. Surprisingly, a branched pattern of clonal evolution is associated with superior clinical outcomes. Finally, several mutations (including NRAS and IDH1) predict drug sensitivity based on their subclonal abundance. Together, these results demonstrate the importance of assessing clonal heterogeneity with implications for prognosis and actionable biomarkers for therapy.