Mapping the cellular biogeography of human bone marrow niches using single-cell transcriptomics and proteomic imaging.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

MM optimization: Proximal distance algorithms, path following, and trust regions.

We briefly review the majorization-minimization (MM) principle and elaborate on the closely related notion of proximal distance algorithms, a generic approach for solving constrained optimization problems via quadratic penalties. We illustrate how the MM and proximal distance principles apply to a variety of problems from statistics, finance, and nonlinear optimization. Drawing from our selected examples, we also sketch a few ideas pertinent to the acceleration of MM algorithms: a) structuring updates around efficient matrix decompositions, b) path following in proximal distance iteration, and c) cubic majorization and its connections to trust region methods. These ideas are put to the test on several numerical examples, but for the sake of brevity, we omit detailed comparisons to competing methods. The current article, which is a mix of review and current contributions, celebrates the MM principle as a powerful framework for designing optimization algorithms and reinterpreting existing ones.

Enrollment Trends Among Patients with Melanoma Brain Metastasis in Active Clinical Trials.

The CNS is a common site for distant metastasis and treatment failure in melanoma patients. This study aimed to evaluate the inclusion rate of patients with melanoma brain metastases (MBM) in prospective clinical trials. 69.3% of trials excluded MBM patients based on their CNS disease. In univariate analysis, trials not employing immunotherapy (p = 0.0174), inclusion of leptomeningeal disease (p < 0.0001) and non-pharmaceutical sponsor trials (p = 0.0461) were more likely to enroll patients with MBM. Thoughtful reconsideration of clinical trial designs is needed to give patients with MBMs access to promising investigational agents and improve outcomes for patients with MBM.

Inferring HIV transmission patterns from viral deep-sequence data via latent typed point processes.

Viral deep-sequencing data play a crucial role toward understanding disease transmission network flows, providing higher resolution compared to standard Sanger sequencing. To more fully utilize these rich data and account for the uncertainties in outcomes from phylogenetic analyses, we propose a spatial Poisson process model to uncover human immunodeficiency virus (HIV) transmission flow patterns at the population level. We represent pairings of individuals with viral sequence data as typed points, with coordinates representing covariates such as gender and age and point types representing the unobserved transmission statuses (linkage and direction). Points are associated with observed scores on the strength of evidence for each transmission status that are obtained through standard deep-sequence phylogenetic analysis. Our method is able to jointly infer the latent transmission statuses for all pairings and the transmission flow surface on the source-recipient covariate space. In contrast to existing methods, our framework does not require preclassification of the transmission statuses of data points, and instead learns them probabilistically through a fully Bayesian inference scheme. By directly modeling continuous spatial processes with smooth densities, our method enjoys significant computational advantages compared to previous methods that rely on discretization of the covariate space. We demonstrate that our framework can capture age structures in HIV transmission at high resolution, bringing valuable insights in a case study on viral deep-sequencing data from Southern Uganda.

Detecting changes in the transmission rate of a stochastic epidemic model.

Throughout the course of an epidemic, the rate at which disease spreads varies with behavioral changes, the emergence of new disease variants, and the introduction of mitigation policies. Estimating such changes in transmission rates can help us better model and predict the dynamics of an epidemic, and provide insight into the efficacy of control and intervention strategies. We present a method for likelihood-based estimation of parameters in the stochastic susceptible-infected-removed model under a time-inhomogeneous transmission rate comprised of piecewise constant components. In doing so, our method simultaneously learns change points in the transmission rate via a Markov chain Monte Carlo algorithm. The method targets the exact model posterior in a difficult missing data setting given only partially observed case counts over time. We validate performance on simulated data before applying our approach to data from an Ebola outbreak in Western Africa and COVID-19 outbreak on a university campus.

WashU Epigenome Browser update 2022.

WashU Epigenome Browser (https://epigenomegateway.wustl.edu/browser/) is a web-based genomic data exploration tool that provides visualization, integration, and analysis of epigenomic datasets. The newly renovated user interface and functions have enabled researchers to engage with the browser and genomic data more efficiently and effectively since 2018. Here, we introduce a new integrated panel design in the browser that allows users to interact with 1D (genomic features), 2D (such as Hi-C), 3D (genome structure), and 4D (time series) data in a single web page. The browser can display three-dimensional chromatin structures with the 3D viewer module. The 4D tracks, called 'Dynamic' tracks, animatedly display time-series data, allowing for a more striking visual impact to identify the gene or genomic region candidates as a function of time. Genomic data, such as annotation features, numerical values, and chromatin interaction data can all be viewed in the dynamic track mode. Imaging data from microscopy experiments can also be displayed in the browser. In addition to software development, we continue to service and expand the data hubs we host for large consortia including 4DN, Roadmap Epigenomics, TaRGET and ENCODE, among others. Our growing user/developer community developed additional track types as plugins, such as qBed and dynseq tracks, which extend the utility of the browser. The browser serves as a foundation for additional genomics platforms including the WashU Virus Genome Browser (for COVID-19 research) and the Comparative Genome Browser. The WashU Epigenome Browser can also be accessed freely through Amazon Web Services at https://epigenomegateway.org/.

Diagnosis, management, and outcomes of immune checkpoint inhibitor induced acute interstitial nephritis: A single-center experience.

BACKGROUND: Immune checkpoint inhibitor (ICI)-associated acute interstitial nephritis (AIN) is a recognized complication of immunotherapy (IO), but literature on its management and outcomes is limited. METHODS: We retrospectively reviewed patients who received ICIs and developed biopsy-proven or clinically-suspected ICI-associated AIN at the University of Virginia Comprehensive Cancer Center from 2012-2023. We analyzed baseline characteristics and clinical outcomes, including treatment interruption and rechallenge rates. Acute kidney injury (AKI) was defined as a ≥ 1.5-fold increase in baseline creatinine under seven days, a two-fold increase above the upper limit of normal, or an increase by ≥0.3 mg/dL. Kidney function returning to within 0.3 mg/dL or less than twice baseline was considered complete (CRc) and partial (PRc) recovery, respectively. RESULTS: We identified 12 cases of ICI-AIN: four by biopsy (33%) and eight (67%) by clinical suspicion. Two patients received anti-CTLA-4 and anti-PD1, six received anti-PD1 alone, and four received chemo-immunotherapy. The majority (58%) of patients developed AIN within the first 5 cycles. Eight patients developed ≥ Grade 3 AKI, and six developed multiple irAEs. ICI was permanently discontinued in seven patients (58%) and temporarily interrupted in four (30%). The CRc and PRc rates were 67% and 8%, respectively. Upon AIN onset, the best disease response was stable disease in five patients, partial response in three, and progressive disease in three. Median overall survival was 4.87 years, and progression-free survival was 1.5 years. CONCLUSIONS: Rechallenge with IO after kidney irAE may be possible in some patients but requires careful evaluation on an individual basis.

Fitting a stochastic model of intensive care occupancy to noisy hospitalization time series during the COVID-19 pandemic.

Intensive care occupancy is an important indicator of health care stress that has been used to guide policy decisions during the COVID-19 pandemic. Toward reliable decision-making as a pandemic progresses, estimating the rates at which patients are admitted to and discharged from hospitals and intensive care units (ICUs) is crucial. Since individual-level hospital data are rarely available to modelers in each geographic locality of interest, it is important to develop tools for inferring these rates from publicly available daily numbers of hospital and ICU beds occupied. We develop such an estimation approach based on an immigration-death process that models fluctuations of ICU occupancy. Our flexible framework allows for immigration and death rates to depend on covariates, such as hospital bed occupancy and daily SARS-CoV-2 test positivity rate, which may drive changes in hospital ICU operations. We demonstrate via simulation studies that the proposed method performs well on noisy time series data and apply our statistical framework to hospitalization data from the University of California, Irvine (UCI) Health and Orange County, California. By introducing a likelihood-based framework where immigration and death rates can vary with covariates, we find, through rigorous model selection, that hospitalization and positivity rates are crucial covariates for modeling ICU stay dynamics and validate our per-patient ICU stay estimates using anonymized patient-level UCI hospital data.

Establishing a rapid assessment service for patients with suspected malignancies for expedited outpatient management.

BACKGROUND: 11% of new cancer diagnoses occur in the emergency department. Historically, these diagnoses disproportionately affect underserved patient populations and are associated with poor outcomes. This is an observational study of the Rapid Assessment Service (RAS) program, which aims to provide timely outpatient follow-up and facilitate a diagnosis for patients discharged from the emergency department with suspected malignancies. METHODS: We performed a retrospective chart review of 176 patients who were discharged from the emergency department with RAS clinic follow up between February 2020 and March 2022. We manually chart reviewed 176 records in order to determine the average time to RAS clinic appointment, average time to diagnosis, and the final diagnosis based on biopsy. RESULTS: 163 of 176 patients (93%) discharged to RAS received reliable follow-up care. 62 of the 176 patients (35%) followed up in the RAS clinic with a mean of 4.6 days. 46 of the 62 patients (74%) who followed up in the RAS clinic were ultimately diagnosed with a new cancer, with a mean time to diagnosis of 13.5 days. The leading new cancer diagnoses included: lung, ovarian, hematologic, head and neck, and renal cancers. CONCLUSIONS: Creating a Rapid Assessment Service facilitated an expedited oncologic work-up and diagnosis in an outpatient setting.

Kikuchi-Fujimoto disease is mediated by an aberrant type I interferon response.

Kikuchi-Fujimoto disease (KFD) is a reactive lymphadenitis of unclear etiology. To understand the pathogenesis of KFD, we performed targeted RNA sequencing of a well-characterized cohort of 15 KFD specimens with 9 non-KFD lymphadenitis controls. Two thousand and three autoimmunity-related genes were evaluated from archived formalin-fixed paraffin-embedded lymph node tissue and analyzed by a bioinformatics approach. Differential expression analysis of KFD cases compared to controls revealed 44 significantly upregulated genes in KFD. Sixty-eight percent of these genes were associated with the type I interferon (IFN) response pathway. Key component of the pathway including nucleic acid sensors, IFN regulatory factors, IFN-induced antiviral proteins, IFN transcription factors, IFN-stimulated genes, and IFN-induced cytokines were significantly upregulated. Unbiased gene expression pathway analysis revealed enrichment of IFN signaling and antiviral pathways in KFD. Protein-protein interaction analysis and a molecular complex detection algorithm identified a densely interacting 15-gene module of type I IFN pathway genes. Apoptosis regulator IFI6 was identified as a key seed gene. Transcription factor target analysis identified enrichment of IFN-response elements and IFN-response factors. T-cell-associated genes were upregulated while myeloid and B-cell-associated genes were downregulated in KFD. CD123+ plasmacytoid dendritic cells (PDCs) and activated T cells were noted in KFD. In conclusion, KFD is mediated by an aberrant type I interferon response that is likely driven by PDCs and T cells.

Transcriptome and unique cytokine microenvironment of Castleman disease.

Castleman disease (CD) represents a group of rare, heterogeneous and poorly understood disorders that share characteristic histopathological features. Unicentric CD (UCD) typically involves a single enlarged lymph node whereas multicentric CD (MCD) involves multiple lymph node stations. To understand the cellular basis of CD, we undertook a multi-platform analysis using targeted RNA sequencing, RNA in-situ hybridization (ISH), and adaptive immune receptor rearrangements (AIRR) profiling of archived tissue from 26 UCD, 14 MCD, and 31 non-CD reactive controls. UCD showed differential expression and upregulation of follicular dendritic cell markers (CXCL13, clusterin), angiogenesis factors (LPL, DLL4), extracellular matrix remodeling factors (TGFß, SKIL, LOXL1, IL-1ß, ADAM33, CLEC4A), complement components (C3, CR2) and germinal center activation markers (ZDHHC2 and BLK) compared to controls. MCD showed upregulation of IL-6 (IL-6ST, OSMR and LIFR), IL-2, plasma cell differentiation (XBP1), FDC marker (CXCL13, clusterin), fibroblastic reticular cell cytokine (CCL21), angiogenesis factor (VEGF), and mTORC1 pathway genes compared to UCD and controls. ISH studies demonstrated that VEGF was increased in the follicular dendritic cell-predominant atretic follicles and the interfollicular macrophages of MCD compared to UCD and controls. IL-6 expression was higher along interfollicular vasculature-associated cells of MCD. Immune repertoire analysis revealed oligoclonal expansions of T-cell populations in MCD cases (2/6) and UCD cases (1/9) that are consistent with antigen-driven T cell activation. The findings highlight the unique genes, pathways and cell types involved in UCD and MCD. We identify potential novel targets in CD that may be harnessed for therapeutics.

Computational tools for assessing gene therapy under branching process models of mutation.

Multitype branching processes are ideal for studying the population dynamics of stem cell populations undergoing mutation accumulation over the years following transplant. In such stochastic models, several quantities are of clinical interest as insertional mutagenesis carries the potential threat of leukemogenesis following gene therapy with autologous stem cell transplantation. In this paper, we develop a three-type branching process model describing accumulations of mutations in a population of stem cells distinguished by their ability for long-term self-renewal. Our outcome of interest is the appearance of a double-mutant cell, which carries a high potential for leukemic transformation. In our model, a single-hit mutation carries a slight proliferative advantage over a wild-type stem cells. We compute marginalized transition probabilities that allow us to capture important quantitative aspects of our model, including the probability of observing a double-hit mutant and relevant moments of a single-hit mutation population over time. We thoroughly explore the model behavior numerically, varying birth rates across the initial sizes and populations of wild type stem cells and single-hit mutants, and compare the probability of observing a double-hit mutant under these conditions. We find that increasing the number of single-mutants over wild-type particles initially present has a large effect on the occurrence of a double-mutant, and that it is relatively safe for single-mutants to be quite proliferative, provided the lentiviral gene addition avoids creating single mutants in the original insertion process. Our approach is broadly applicable to an important set of questions in cancer modeling and other population processes involving multiple stages, compartments, or types.

Surgical Tray Optimization: a Quality Improvement Initiative that Reduces Operating Room Costs.

Surgical trays contain unused instruments which generate wasted resources from unnecessary reprocessing/replacement costs. We implemented a quality improvement initiative to optimize surgical trays for common otolaryngology procedures, and examined the impact on costs, operating room (OR) efficiency, and patient safety.We studied five common otolaryngology procedures over a 10-month period at a single community hospital. We compared pre- and post-intervention outcome measures including instrument utilization, tray set up time, tray rebuilding time, and balancing measures (operative time, instrument recall, patient safety). We estimated cost-savings from an institutional perspective over 1- and 10-year time horizons. Costs were expressed in 2017 Canadian dollars and modeled as a function of surgical volume, labor costs, instrument depreciation, and indirect costs.A total of 238 procedures by six surgeons were observed. At baseline, only 35% of instruments were utilized. We achieved an average instrument reduction of 26%, yielding 1-year cost savings of $9,010 CDN and 10-year cost savings of $69,576 CDN. Tray optimization reduced average OR tray setup time by 2.5 ± 0.4 min (p = 0.03) and average tray rebuilding time by 1.4 ± 0.2 min (p = 0.06). There was minimal impact on balancing measures such as OR time, stakeholder perception of patient safety and trainee education, and only a single case of instrument recall.Surgical tray optimization is a simple, effective, and scalable strategy for reducing costs and improving OR efficiency without compromising patient safety.

Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking.

Devoid of all known canonical actin-binding proteins, the prevalent parasite Giardia lamblia uses an alternative mechanism for cytokinesis. Unique aspects of this mechanism can potentially be leveraged for therapeutic development. Here, live-cell imaging methods were developed for Giardia to establish division kinetics and the core division machinery. Surprisingly, Giardia cytokinesis occurred with a median time that is ∼60 times faster than mammalian cells. In contrast to cells that use a contractile ring, actin was not concentrated in the furrow and was not directly required for furrow progression. Live-cell imaging and morpholino depletion of axonemal Paralyzed Flagella 16 indicated that flagella-based forces initiated daughter cell separation and provided a source for membrane tension. Inhibition of membrane partitioning blocked furrow progression, indicating a requirement for membrane trafficking to support furrow advancement. Rab11 was found to load onto the intracytoplasmic axonemes late in mitosis and to accumulate near the ends of nascent axonemes. These developing axonemes were positioned to coordinate trafficking into the furrow and mark the center of the cell in lieu of a midbody/phragmoplast. We show that flagella motility, Rab11, and actin coordination are necessary for proper abscission. Organisms representing three of the five eukaryotic supergroups lack myosin II of the actomyosin contractile ring. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II and additionally implicate the broad use of membrane tension as a mechanism to drive abscission.

Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants.

Autologous transplantation of hematopoietic stem and progenitor cells lentivirally labeled with unique oligonucleotide barcodes flanked by sequencing primer targets enables quantitative assessment of the self-renewal and differentiation patterns of these cells in a myeloablative rhesus macaque model. Compared with other approaches to clonal tracking, this approach is highly quantitative and reproducible. We documented stable multipotent long-term hematopoietic clonal output of monocytes, granulocytes, B cells, and T cells from a polyclonal pool of hematopoietic stem and progenitor cells in 4 macaques observed for up to 49 months posttransplantation. A broad range of clonal behaviors characterized by contribution level and biases toward certain cell types were extremely stable over time. Correlations between granulocyte and monocyte clonalities were greatest, followed by correlations between these cell types and B cells. We also detected quantitative expansion of T cell-biased clones consistent with an adaptive immune response. In contrast to recent data from a nonquantitative murine model, there was little evidence for clonal succession after initial hematopoietic reconstitution. These findings have important implications for human hematopoiesis, given the similarities between macaque and human physiologies.

Mechanisms of neuroprotection against ischemic insult by stress-inducible phosphoprotein-1/prion protein complex.

Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrPC ) and activating α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1-h oxygen-glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrPC . Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrPC and α7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrPC /STI1 engagement and its subsequent downstream signaling cascades involving α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults.

Birth/birth-death processes and their computable transition probabilities with biological applications.

Birth-death processes track the size of a univariate population, but many biological systems involve interaction between populations, necessitating models for two or more populations simultaneously. A lack of efficient methods for evaluating finite-time transition probabilities of bivariate processes, however, has restricted statistical inference in these models. Researchers rely on computationally expensive methods such as matrix exponentiation or Monte Carlo approximation, restricting likelihood-based inference to small systems, or indirect methods such as approximate Bayesian computation. In this paper, we introduce the birth/birth-death process, a tractable bivariate extension of the birth-death process, where rates are allowed to be nonlinear. We develop an efficient algorithm to calculate its transition probabilities using a continued fraction representation of their Laplace transforms. Next, we identify several exemplary models arising in molecular epidemiology, macro-parasite evolution, and infectious disease modeling that fall within this class, and demonstrate advantages of our proposed method over existing approaches to inference in these models. Notably, the ubiquitous stochastic susceptible-infectious-removed (SIR) model falls within this class, and we emphasize that computable transition probabilities newly enable direct inference of parameters in the SIR model. We also propose a very fast method for approximating the transition probabilities under the SIR model via a novel branching process simplification, and compare it to the continued fraction representation method with application to the 17th century plague in Eyam. Although the two methods produce similar maximum a posteriori estimates, the branching process approximation fails to capture the correlation structure in the joint posterior distribution.

Discovery of Small Molecule Ligands for MALAT1 by Tuning an RNA-Binding Scaffold.

Structural studies of the 3'-end of the oncogenic long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) confirmed a unique triple-helix structure. This structure enables accumulation of the transcript, and high levels of MALAT1 are found in several cancers. Here, we synthesize a small molecule library based on an RNA-binding scaffold, diphenylfuran (DPF), screen it against a variety of nucleic acid constructs, and demonstrate for the first time that the MALAT1 triple helix can be selectively targeted with small molecules. Computational analysis revealed a trend between subunit positioning and composition on DPF shape and intramolecular interactions, which in turn generally correlated with selectivity and binding strengths. This work thus provides design strategies toward chemical probe development for the MALAT1 triple helix and suggests that comprehensive analyses of RNA-focused libraries can generate insights into selective RNA recognition.

Cholinergic Regulation of hnRNPA2/B1 Translation by M1 Muscarinic Receptors.

UNLABELLED: Cholinergic vulnerability, characterized by loss of acetylcholine (ACh), is one of the hallmarks of Alzheimer's disease (AD). Previous work has suggested that decreased ACh activity in AD may contribute to pathological changes through global alterations in alternative splicing. This occurs, at least partially, via the regulation of the expression of a critical protein family in RNA processing, heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins. These proteins regulate several steps of RNA metabolism, including alternative splicing, RNA trafficking, miRNA export, and gene expression, providing multilevel surveillance in RNA functions. To investigate the mechanism by which cholinergic tone regulates hnRNPA2/B1 expression, we used a combination of genetic mouse models and in vivo and in vitro techniques. Decreasing cholinergic tone reduced levels of hnRNPA2/B1, whereas increasing cholinergic signaling in vivo increased expression of hnRNPA2/B1. This effect was not due to decreased hnRNPA2/B1 mRNA expression, increased aggregation, or degradation of the protein, but rather to decreased mRNA translation by nonsense-mediated decay regulation of translation. Cell culture and knock-out mice experiments demonstrated that M1 muscarinic signaling is critical for cholinergic control of hnRNPA2/B1 protein levels. Our experiments suggest an intricate regulation of hnRNPA2/B1 levels by cholinergic activity that interferes with alternative splicing in targeted neurons mimicking deficits found in AD. SIGNIFICANCE STATEMENT: In Alzheimer's disease, degeneration of basal forebrain cholinergic neurons is an early event. These neurons communicate with target cells and regulate their long-term activity by poorly understood mechanisms. Recently, the splicing factor hnRNPA2/B, which is decreased in Alzheimer's disease, was implicated as a potential mediator of long-term cholinergic regulation. Here, we demonstrate a mechanism by which cholinergic signaling controls the translation of hnRNPA2/B1 mRNA by activation of M1 muscarinic type receptors. Loss of cholinergic activity can have profound effects in target cells by modulating hnRNPA2/B1 levels.

Determining Patient Preferences for Indeterminate Thyroid Nodules: Observation, Surgery or Molecular Tests.

BACKGROUND: Gene-expression classifiers (GEC) and genetic mutation panels (GMP) have been shown to improve preoperative diagnostic evaluations of indeterminate thyroid nodules. Despite the improvement, uncertainty regarding the proper management exists. Patient preferences may better inform the management of these indeterminate thyroid nodules. METHODS: Hypothetical scenarios were administered to two groups of patients: those with previous FNA-confirmed indeterminate thyroid nodules (Group A, n = 50) and those presenting to a general otolaryngology clinic for other reasons (Group B, n = 50). We evaluated patient preferences for surgery, observation and the use of molecular tests while varying the risk of malignancy, cost and diagnostic properties of the tests. RESULTS: The mean threshold for choosing surgery over observation was a 38.6% risk of malignancy on FNA. When offered either GEC, GMP or both (with their inherent imperfect diagnostic properties) in addition to the indeterminate FNA, 85.0% of respondents picked at least one of the molecular tests over either observation or surgery if the test(s) were free of charge. However, only 51.7% of respondents chose at least one of the tests when asked to pay the current cost of the test(s) (p < 0.001). On multivariable analysis, sex, the presence of an indeterminate FNA diagnosis and income level significantly predicted the desire to proceed with a molecular test above standard management. CONCLUSION: Patient preferences for thyroid nodule management are dependent on the risk of malignancy, prognosis of cancer and costs. Patients prefer molecular tests over standard management with indeterminate thyroid nodules, but the costs of the test(s) reduce the desire.