The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment.

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.

Predictive Value of Diaphragm Muscle Ultrasound for Ventilator Weaning Outcomes After Cervical Spinal Cord Injury: A Retrospective Case Series.

OBJECTIVES: Neuromuscular respiratory failure after cervical spinal cord injury (cSCI) can lead to dependence on an invasive mechanical ventilator. Ventilator-free breathing after cSCI is associated with improved morbidity, mortality, and quality of life. We investigated the use of diaphragm muscle ultrasound to predict ventilator weaning outcomes after cSCI. METHODS: This is a retrospective case series conducted at a university-affiliated freestanding inpatient rehabilitation facility. We identified patients with cSCI who had a tracheostomy and were dependent on an invasive mechanical ventilator at the time of admission to inpatient rehabilitation. A diaphragm muscle ultrasound was performed, which included measurements of the thickness of the diaphragm and a calculation of the thickening ratio (TR), which reflects diaphragm muscle contraction. The primary outcome measure was the need for mechanical ventilation at time of discharge from the inpatient rehabilitation facility. Successful ventilator weaning was defined as either daytime or full 24-hour ventilator-free breathing. RESULTS: Of the 21 patients enrolled, 11 (52%) were able to wean successfully (partially or fully) from the ventilator. Of the ultrasound measurements that were taken, the TR was the optimal predictor for ventilator weaning outcomes. A threshold of TR ≥ 1.2 as the maximum hemidiaphragm measurement had a sensitivity of 1.0 and specificity of 0.90 for predicting ventilator weaning. CONCLUSION: Normal diaphragm contractility (TR ≥ 1.2) as determined by diaphragm muscle ultrasound is a strong positive predictor for successful ventilator weaning in patients with cSCI. Utilizing diaphragm ultrasound, rehabilitation physicians can set precision rehabilitation goals regarding ventilator weaning for inpatients with respiratory failure after cSCI, potentially improving both outcomes and quality of life.

The virtual pediatric perioperative home, experience at a major metropolitan safety net hospital.

INTRODUCTION: Successes from anesthesiologist-led perioperative surgical homes in the adult patient population have inspired similar initiatives by pediatric hospitals. Typically the care coordination for these perioperative homes is run through hospital-funded, on-site, preanesthesia clinics. Preliminary data from pediatric perioperative homes have shown promising results in improved patient outcomes and decreased length of hospital stay. The majority of pediatric surgeries within the country are performed in nonpediatric hospitals. Such centers may not have the infrastructure or financial resources for a freestanding pediatric preanesthesia clinic. Faced with this situation at the largest safety net hospital in New England, the authors present their experience designing and implementing a "Virtual Pediatric Perioperative Home," a telemedicine-based triage and preanesthetic optimization for pediatric patients at Boston Medical Center, Boston, MA. METHODS: A retrospective chart review of all pediatric anesthesia cases at Boston Medical Center from February 1, 2019, to January 31, 2020, as well as the number of pediatric cases canceled or postponed on the day of surgery for any reason during the same time period was conducted. RESULTS: From February 1, 2019, to January 31, 2020, 1546 anesthetics were performed in children 18 years and under. Of those, 63 were designated as emergent and hence excluded from our analysis. 153 of the total 1483 (9.4%) of nonemergent bookings were canceled or postponed on the day of surgery. This represented a marked decline from our previous year's 13.7% same-day cancellation rate for pediatric patients. The most common reason for case cancellations (41.8%) was acute illness. Cancellation rates varied from month to month, with the highest cancellation rate of the year in September 2019 (18.8%). The departments of Podiatry and Gastroenterology represented the highest cancellation rates as a denominator of their case volumes, 15.4% and 15.2%, respectively. Younger children had 2.4 times the odds (95% CI: 1.720, 3.4) of cancellation compared to older children. DISCUSSION: The virtual pediatric perioperative home (VPPH) may benefit quality of care while decreasing costs to pediatric patients, families, and hospital systems. While direct financial gains may be difficult to demonstrate, the VPPH has the potential to reduce OR delays and same day cancellations related to questions of medical optimization. In the context of a socioeconomically disadvantaged patient population, our VPPH's team of subspecialists created inroads for at risk children to establish or reestablish care for their comorbidities, while collaboration with the Department of Children and Families further streamlined communication and consent for pediatric patients in foster care. CONCLUSIONS: The authors describe the design and successful implementation of a telemedicine-based pediatric preanesthesia triage and medical optimization service at a large safety net hospital. By creating a communication network of pediatric subspecialists, the anesthesiologists were able to, at minimal institutional cost, coordinate care for children with a variety of comorbidities leading up to the day of surgery. This yielded a 9.4% same day cancellation rate in a complex, socioeconomically disadvantaged pediatric patient population at a general hospital.

CIPR: a web-based R/shiny app and R package to annotate cell clusters in single cell RNA sequencing experiments.

BACKGROUND: Single cell RNA sequencing (scRNAseq) has provided invaluable insights into cellular heterogeneity and functional states in health and disease. During the analysis of scRNAseq data, annotating the biological identity of cell clusters is an important step before downstream analyses and it remains technically challenging. The current solutions for annotating single cell clusters generally lack a graphical user interface, can be computationally intensive or have a limited scope. On the other hand, manually annotating single cell clusters by examining the expression of marker genes can be subjective and labor-intensive. To improve the quality and efficiency of annotating cell clusters in scRNAseq data, we present a web-based R/Shiny app and R package, Cluster Identity PRedictor (CIPR), which provides a graphical user interface to quickly score gene expression profiles of unknown cell clusters against mouse or human references, or a custom dataset provided by the user. CIPR can be easily integrated into the current pipelines to facilitate scRNAseq data analysis. RESULTS: CIPR employs multiple approaches for calculating the identity score at the cluster level and can accept inputs generated by popular scRNAseq analysis software. CIPR provides 2 mouse and 5 human reference datasets, and its pipeline allows inter-species comparisons and the ability to upload a custom reference dataset for specialized studies. The option to filter out lowly variable genes and to exclude irrelevant reference cell subsets from the analysis can improve the discriminatory power of CIPR suggesting that it can be tailored to different experimental contexts. Benchmarking CIPR against existing functionally similar software revealed that our algorithm is less computationally demanding, it performs significantly faster and provides accurate predictions for multiple cell clusters in a scRNAseq experiment involving tumor-infiltrating immune cells. CONCLUSIONS: CIPR facilitates scRNAseq data analysis by annotating unknown cell clusters in an objective and efficient manner. Platform independence owing to Shiny framework and the requirement for a minimal programming experience allows this software to be used by researchers from different backgrounds. CIPR can accurately predict the identity of a variety of cell clusters and can be used in various experimental contexts across a broad spectrum of research areas.

Association of Anesthesia Type with Outcomes after Outpatient Brachiocephalic Arteriovenous Fistula Creation.

BACKGROUND: Brachiocephalic arteriovenous fistulas (BCFs) are commonly placed in outpatient settings. The impact of general anesthesia (GA), regional anesthesia (RA), or local anesthesia (LA) on perioperative recovery and fistula maturation/patency after outpatient BCF creations is unknown. We evaluated whether outcomes of outpatient BCF creations vary based on anesthesia modality. METHODS: The Vascular Quality Initiative (2011-2018) national database was queried for outpatient BCF creations. Anesthesia modalities included GA, RA, and LA. Perioperative, 3-month, and 1-year outcomes were compared between GA versus RA/LA anesthesia types. RESULTS: Among 3,527 outpatient BCF creations, anesthesia types were GA in 1,043 (29.6%), RA in 1,150 (32.6%), and LA in 1,334 (37.8%). Patients receiving GA were more often younger, obese, Medicaid recipients, without coronary artery disease, and treated in non-office-based settings (P < 0.05 for all). GA compared with RA/LA cohorts were more often admitted postoperatively (5.3% vs. 2.4%, P < 0.001) but had similar rates of thirty-day mortality (0.9 vs. 0.6%, P = 0.39). 3-month access utilization for hemodialysis was lower in GA than in RA/LA cohorts (12.6% vs. 23.6%, P < 0.001). The Kaplan-Meier analysis showed that GA and RA/LA cohorts had similar 1-year primary access occlusion-free survival (43.6% vs. 47.1%, P = 0.24) and endovascular/open reintervention-free survival (57.2% vs. 57.6%, P = 0.98). On multivariable analysis, GA compared with RA/LA use was independently associated with increased postoperative admission (odds ratio [OR]: 1.7, 95% confidence interval [CI]: 1.08-2.67, P = 0.02) and decreased 3-month access utilization (OR: 0.39, 95% CI: 0.25-0.61, P < 0.001) but had similar 1-year access occlusion (hazard ratio [HR]: 1.09, 95% CI: 0.9-1.32, P = 0.36) and reintervention (HR: 1.02, 95% CI: 0.82-1.26, P = 0.88). On subgroup analysis of the RA/LA cohort, RA compared with LA was associated with increased 3-month access utilization (OR: 1.6, 95% CI: 1.01-2.5; P = 0.04) and 1-year access reintervention (HR: 1.46, 95% CI: 1.12-1.89), but had similar 1-year access occlusion (HR: 1.2, 95% CI: 0.95-1.51, P = 0.13). CONCLUSIONS: Compared with RA/LA use, GA use in patients undergoing outpatient BCF creations was associated with increased hospital admissions, decreased access utilization at 3 months, and similar 1-year access occlusion and reintervention. RA/LA is preferable to expedite recovery and access utilization.

A phase 2 study of lenalidomide, rituximab, cyclophosphamide, and dexamethasone (LR-CD) for untreated low-grade non-Hodgkin lymphoma requiring therapy.

Patients with indolent non-Hodgkin lymphoma (NHL) have multiple treatment options yet there is no consensus as to the best initial therapy. Lenalidomide, an immunomodulatory agent, has single agent activity in relapsed lymphoma. This trial was conducted to assess feasibility, efficacy, and safety of adding lenalidomide to rituximab, cyclophosphamide, and dexamethasone (LR-CD) in untreated indolent NHL patients requiring therapy. This was a single institution phase II trial. Treatment consisted of IV rituximab 375 mg/m2 day 1; oral lenalidomide 20 mg days 1-21; cyclophosphamide 250 mg/m2 days 1, 8, and 15; and dexamethasone 40 mg days 1, 8, 15, and 22 of a 28-day cycle. Treatment continued 2 cycles beyond best response for a maximum of 12 cycles without rituximab maintenance. Thirty-three patients were treated. Median age was 68 (43-83 years). 39% had stage IV disease. Histologic subtypes included 8 follicular lymphoma (FL), 7 marginal zone lymphoma (MZL) (1 splenic, 2 extranodal, and 4 nodal), 15 Waldenström's macroglobulinemia (WM), 1 lymphoplasmacytic lymphoma, 1 small lymphocytic lymphoma, and 1 low-grade B-cell lymphoma with plasmacytic differentiation (unable to be classified better as MZL or LPL). Hematologic toxicity was the most common adverse event. Median time of follow-up was 23.4 months (range 1.8-50.9). The overall response rate was 87.9%, with 30.3% complete response. The median duration of response was 38.7 months. The median progression free survival was 39.7 months, while median overall survival (OS) has not yet been reached. Lenalidomide can be safely added to a simple regimen of rituximab, oral cyclophosphamide, and dexamethasone and is an effective combination as initial therapy for low-grade B-cell NHL.

Massifquant: open-source Kalman filter-based XC-MS isotope trace feature detection.

MOTIVATION: Isotope trace (IT) detection is a fundamental step for liquid or gas chromatography mass spectrometry (XC-MS) data analysis that faces a multitude of technical challenges on complex samples. The Kalman filter (KF) application to IT detection addresses some of these challenges; it discriminates closely eluting ITs in the m/z dimension, flexibly handles heteroscedastic m/z variances and does not bin the m/z axis. Yet, the behavior of this KF application has not been fully characterized, as no cost-free open-source implementation exists and incomplete evaluation standards for IT detection persist. RESULTS: Massifquant is an open-source solution for KF IT detection that has been subjected to novel and rigorous methods of performance evaluation. The presented evaluation with accompanying annotations and optimization guide sets a new standard for comparative IT detection. Compared with centWave, matchedFilter and MZMine2-alternative IT detection engines-Massifquant detected more true ITs in a real LC-MS complex sample, especially low-intensity ITs. It also offers competitive specificity and equally effective quantitation accuracy. AVAILABILITY AND IMPLEMENTATION: Massifquant is integrated into XCMS with GPL license ≥ 2.0 and hosted by Bioconductor: http://bioconductor.org. Annotation data are archived at http://hdl.lib.byu.edu/1877/3232. Parameter optimization code and documentation is hosted at https://github.com/topherconley/optimize-it.

Regulation of Zn and Fe transporters by the GPC1 gene during early wheat monocarpic senescence.

BACKGROUND: During wheat senescence, leaf components are degraded in a coordinated manner, releasing amino acids and micronutrients which are subsequently transported to the developing grain. We have previously shown that the simultaneous downregulation of Grain Protein Content (GPC) transcription factors, GPC1 and GPC2, greatly delays senescence and disrupts nutrient remobilization, and therefore provide a valuable entry point to identify genes involved in micronutrient transport to the wheat grain. RESULTS: We generated loss-of-function mutations for GPC1 and GPC2 in tetraploid wheat and showed in field trials that gpc1 mutants exhibit significant delays in senescence and reductions in grain Zn and Fe content, but that mutations in GPC2 had no significant effect on these traits. An RNA-seq study of these mutants at different time points showed a larger proportion of senescence-regulated genes among the GPC1 (64%) than among the GPC2 (37%) regulated genes. Combined, the two GPC genes regulate a subset (21.2%) of the senescence-regulated genes, 76.1% of which are upregulated at 12 days after anthesis, before the appearance of any visible signs of senescence. Taken together, these results demonstrate that GPC1 is a key regulator of nutrient remobilization which acts predominantly during the early stages of senescence. Genes upregulated at this stage include transporters from the ZIP and YSL gene families, which facilitate Zn and Fe export from the cytoplasm to the phloem, and genes involved in the biosynthesis of chelators that facilitate the phloem-based transport of these nutrients to the grains. CONCLUSIONS: This study provides an overview of the transport mechanisms activated in the wheat flag leaf during monocarpic senescence. It also identifies promising targets to improve nutrient remobilization to the wheat grain, which can help mitigate Zn and Fe deficiencies that afflict many regions of the developing world.

Racial and Ethnic Disparities in Situational Judgment Testing Among Applicants to an Anesthesiology Residency Program.

Background The Computer-Based Assessment for Sampling Personal Characteristics (CASPer) is a situational judgment test (SJT) that assesses noncognitive skills like professionalism, communication, and empathy. There are no reports of the effects of race/ethnicity and sex on CASPer scores among residency applicants. Objective We examined the effects of race/ethnicity, sex, and United States vs international medical school attendance on CASPer performance. Methods Our anesthesiology residency program required all applicants for the 2021-2022 Match cycle to complete an online video and text-based SJT (CASPer). We compared these results, reported as z-scores, with self-identified race/ethnicity, sex, United States vs international medical school attendance, and United States Medical Licensing Examination (USMLE) Step 1 scores. Results Of the 1245 applicants who completed CASPer, 783 identified as male. The racial/ethnic distribution was 512 White, 412 Asian, 106 Black, 126 Hispanic, and 89 Other/No Answer. CASPer z-scores did not differ by sex. White candidates scored higher than Black (0.18 vs -0.57, P<.001) and Hispanic (0.18 vs -0.52, P<.001) candidates. Applicants attending US medical schools scored higher than those attending international medical schools (z-scores: 0.15 vs -0.68, P<.001). There was no correlation between CASPer z-scores and USMLE Step 1 scores. Conclusions Our results suggest that CASPer scores favor White applicants over Black and Hispanic ones and applicants attending US medical schools over those attending international medical schools.

Hemophagocytic Lymphohistiocytosis Secondary to Disseminated Histoplasmosis in Rheumatologic Disease.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) was originally described in pediatric patients presenting with fever, hepatosplenomegaly, and blood cell abnormalities. Later, HLH was recognized to occur in adults, often associated with hematologic malignancies or serious infections. CONCLUSION: Patients presenting with HLH are critically ill, and rapid diagnosis is key. In adults, the search for the trigger must begin promptly as time to diagnosis effects survival. The underlying trigger in our patients was Histoplasma capsulatum infection, which is rare in the southwestern United States. Prompt diagnosis led to recovery in one patient, while the other did not survive.

Concurrent Liver and Mesentery Primary Extraosseous Multiple Myeloma on 18F-FDG PET/CT.

ABSTRACT: Extramedullary multiple myeloma (EMM) is a subset of multiple myeloma with a poor prognosis. We report a rare case with biopsy-proven concurrent liver and mesentery primary EMM at the time of initial staging after serologic diagnosis of multiple myeloma. 18F-FDG PET/CT is valuable in detection of EMM when the patient has no osseous lesions and a negative bone marrow biopsy.

Treatment Options for COVID-19-Related Guillain-Barré Syndrome: A Systematic Review of Literature.

BACKGROUND: Central nervous system complications are reported in an increasing number of patients with Coronavirus Disease 2019 (COVID-19). COVID-19-related Guillain-Barré syndrome (GBS) is of particular importance given its association with higher mortality rates and prolonged respiratory failure. REVIEW SUMMARY: We conducted a systematic review of published cases for COVID-19-related GBS, and provide a summary of clinical management strategies for these cases. Sixty-three studies, including 86 patients, were included. Seventy-six cases with reported outcome data were eligible for the outcome analysis. Ninety-nine percent of patients were diagnosed with COVID-19 before diagnosis of GBS (median: 14 d prior, interquartile range: 7 to 20). Intravenous immunotherapy (intravenous immunoglobulin: 0.4 g/kg/d for 5 d) was the most frequently used treatment approach. The review indicated that the outcome was not favorable in 26% of cases (persistent neurological deficits). A mortality rate of 3.5% was observed in patients with COVID-19-related GBS. CONCLUSIONS: Although evidence to support specific treatments is lacking, clinicians should consider the benefits of immunotherapy and plasma exchange in addition to the standard antimicrobial and supportive therapies for patients who meet the diagnostic criteria for acute sensory and motor polyradiculoneuritis. Intravenous immunoglobulin treatment alone is not shown to result in improved outcomes or mortality. More extensive studies aimed at exploring the neurological manifestations and complications of COVID-19 and distinctive treatment options for COVID-19-related GBS are warranted.

The stochastic nature of errors in next-generation sequencing of circulating cell-free DNA.

Challenges with distinguishing circulating tumor DNA (ctDNA) from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Here we show early and random PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding, removal of artifacts due to clonal hematopoiesis of indeterminate potential, and suppression of patterned errors. We also demonstrate sample duplicates are necessary to eliminate the stochastic noise associated with NGS. Integration of sample duplicates into NGS analytics may broaden ctDNA applications by removing NGS-related errors that confound identification of true very low frequency variants during searches for ctDNA without a priori knowledge of specific mutations to target.

MYC Drives Temporal Evolution of Small Cell Lung Cancer Subtypes by Reprogramming Neuroendocrine Fate.

Small cell lung cancer (SCLC) is a neuroendocrine tumor treated clinically as a single disease with poor outcomes. Distinct SCLC molecular subtypes have been defined based on expression of ASCL1, NEUROD1, POU2F3, or YAP1. Here, we use mouse and human models with a time-series single-cell transcriptome analysis to reveal that MYC drives dynamic evolution of SCLC subtypes. In neuroendocrine cells, MYC activates Notch to dedifferentiate tumor cells, promoting a temporal shift in SCLC from ASCL1+ to NEUROD1+ to YAP1+ states. MYC alternatively promotes POU2F3+ tumors from a distinct cell type. Human SCLC exhibits intratumoral subtype heterogeneity, suggesting that this dynamic evolution occurs in patient tumors. These findings suggest that genetics, cell of origin, and tumor cell plasticity determine SCLC subtype.

Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. EXPERIMENTAL DESIGN: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. RESULTS: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. CONCLUSIONS: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

Histopathologic Evaluation of Flexor Tenosynovium in Recurrent Carpal Tunnel Syndrome.

BACKGROUND: The authors' purpose was to evaluate the histopathology of flexor tenosynovium in true, idiopathic recurrent carpal tunnel syndrome for the presence of abnormal inflammatory or pathologic findings that might explain causation or that differ from those previously described for primary, idiopathic carpal tunnel syndrome. METHODS: Thirty-five patients (19 women and 16 men; mean age, 72 years) underwent open revision carpal tunnel release a mean 13 years (range, 0.5 to 30 years) after primary carpal tunnel release. Recurrence was confirmed by recurrent symptoms, positive provocative tests, and electrodiagnostic testing. All patients underwent tenosynovial biopsy, including Congo red staining for amyloid. RESULTS: Histopathologic findings demonstrated noninflammatory, fibrous connective tissue in 31 of 35 patients (89 percent); and mild, chronic inflammation (without granulomas) in four of 35 patients (11 percent). Nine of 35 patients (26 percent) had positive results for amyloid, with a statistically higher incidence in men (p = 0.03) and advanced age (p = 0.02). Subtyping performed in eight of nine amyloid-positive specimens confirmed seven cases of transthyretin-type amyloid typically seen in localized (senile) amyloidosis and one case of light-chain amyloid in a patient who was subsequently diagnosed with myeloma. CONCLUSIONS: Flexor tenosynovium in patients with recurrent carpal tunnel syndrome does not appear to be substantially different histologically from that previously described in primary idiopathic carpal tunnel syndrome, although a slightly higher prevalence of amyloid was seen in this group (especially older men). No patients have developed systemic amyloidosis. Routine biopsy of tenosynovium in idiopathic, recurrent carpal tunnel syndrome is unnecessary.

Heterogeneous beta-catenin activation is sufficient to cause hepatocellular carcinoma in zebrafish.

Up to 41% of hepatocellular carcinomas (HCCs) result from activating mutations in the CTNNB1 gene encoding ß-catenin. HCC-associated CTNNB1 mutations stabilize the ß-catenin protein, leading to nuclear and/or cytoplasmic localization of ß-catenin and downstream activation of Wnt target genes. In patient HCC samples, ß-catenin nuclear and cytoplasmic localization are typically patchy, even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ß-catenin activation are not well understood. To define mechanisms of ß-catenin-driven HCC initiation, we generated a Cre-lox system that enabled switching on activated ß-catenin in (1) a small number of hepatocytes in early development; or (2) the majority of hepatocytes in later development or adulthood. We discovered that switching on activated ß-catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/ß-catenin signaling heterogeneity later in hepatocarcinogenesis, we performed RNA-seq analysis of zebrafish ß-catenin-driven HCC. At the single-cell level, 2.9% to 15.2% of hepatocytes from zebrafish ß-catenin-driven HCC expressed two or more of the Wnt target genes axin2, mtor, glula, myca and wif1, indicating focal activation of Wnt signaling in established tumors. Thus, heterogeneous ß-catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis.

Decreased warfarin sensitivity among patients treated with elbasvir and grazoprevir for hepatitis C infection.

PURPOSE: We previously reported an interaction with warfarin anticoagulation when initiating treatment with direct-acting antiviral agents for hepatitis C infection. A decreased warfarin sensitivity led to subtherapeutic anticoagulation. To study this interaction further, we expanded our research to include patients treated with the combination of elbasvir and grazoprevir concurrent with warfarin anticoagulation and investigated changes in warfarin sensitivity during and after treatment. METHODS: Using electronic health records of the Veterans Health Administration, patients starting treatment with elbasvir-grazoprevir for hepatitis C infection concurrent with warfarin anticoagulation were identified. Inclusion required stable warfarin anticoagulation prior to 12 weeks of treatment with elbasvir-grazoprevir. A warfarin sensitivity index (WSI) was calculated at the start of treatment, after 12 weeks after treatment, and at the end of treatment. The primary endpoint was the difference in WSI from pre- to end-treatment. The secondary endpoint was the WSI difference from before treatment to Changes in International Normalized Ratio, warfarin doses, and time in therapeutic range were measured. RESULTS: In the final sample of 43 patients, the mean WSI decreased during treatment from 0.53 to 0.40, or 25.2%. After treatment, the mean WSI rose to 0.51. Although the mean weekly warfarin dose increased from 40.3 to 44.6 mg during treatment, the mean International Normalized Ratio decreased from 2.40 to 1.96, recovering to 2.59 after treatment. The time spent in therapeutic range decreased from 74.1% before treatment to 39.8% during treatment and back to 64.9% 12 weeks posttreatment. CONCLUSION: When elbasvir-grazoprevir was added to stable warfarin anticoagulation, warfarin sensitivity decreased significantly during treatment and returned to baseline after treatment.