An indocyanine green-based liquid biopsy test for circulating tumor cells for pediatric liver cancer.

BACKGROUND: Hepatoblastoma and HCC are the most common malignant hepatocellular tumors seen in children. The aim of this study was to develop a liquid biopsy test for circulating tumor cells (CTCs) for these tumors that would be less invasive and provide real-time information about tumor response to therapy. METHODS: For this test, we utilized indocyanine green (ICG), a far-red fluorescent dye used clinically to identify malignant liver cells during surgery. We assessed ICG accumulation in cell lines using fluorescence microscopy and flow cytometry. For our CTC test, we developed a panel of liver tumor-specific markers, including ICG, Glypican-3, and DAPI, and tested it with cell lines and noncancer control blood samples. We then used this panel to analyze whole-blood samples for CTC burden with a cohort of 15 patients with hepatoblastoma and HCC and correlated with patient characteristics and outcomes. RESULTS: We showed that ICG accumulation is specific to liver cancer cells, compared to nonmalignant liver cells, non-liver solid tumor cells, and other nonmalignant cells, and can be used to identify liver tumor cells in a mixed population of cells. Experiments with the ICG/Glypican-3/DAPI panel showed that it specifically tagged malignant liver cells. Using patient samples, we found that CTC burden from sequential blood samples from the same patients mirrored the patients' responses to therapy. CONCLUSIONS: Our novel ICG-based liquid biopsy test for CTCs can be used to specifically detect and quantify CTCs in the blood of pediatric patients with liver cancer.

Histopathologic and immunophenotypic characterization of patient-derived pediatric malignant hepatocellular tumor xenografts (PDXs).

Advances in targeted therapies for pediatric hepatocellular tumors have been limited due to a paucity of clinically relevant models. Establishment and validation of intrahepatic patient-derived xenograft (PDX) models would help bridging this gap. The aim of this study is to compare the histomorphologic and immunophenotypic fidelity of patient tumors and their corresponding intrahepatic PDX models. Murine PDX models were established by intrahepatic implantation of patient tumors. Pathology slides from both patients and their corresponding PDX models were reviewed and quantitatively assessed for various histologic components and immunophenotypic markers. Ten PDX models were successfully established from nine patients with pre- (n=3) and post- (n=6) chemotherapy samples; diagnosed of hepatoblastoma (n=8) and hepatocellular neoplasm, not otherwise specified (n=1). Two of nine (22.2%) patients showed ≥75% fetal component; however, the corresponding PDX models did not maintain this fetal differentiation. High grade histology was seen in three patients (33.3%) and overrepresented in six PDX models (60%). Within the subset of three PDXs that were further characterized, significant IHC concordance was seen in all 3 models for CK7, CK19, Ki-67, and p53; and 2 of 3 models for Sox9 and Beta-catenin. GPC-3 and GS showed variable to moderate concordance, while Hepar was the least concordant. Our study shows that in general, the PDX models appear to represent the higher-grade component of the original tumor and show significant concordance for Ki-67, making them appropriate tools for testing new therapies for the most aggressive, therapy-resistant tumors.

A novel treatment strategy utilizing panobinostat for high-risk and treatment-refractory hepatoblastoma.

BACKGROUND & AIMS: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To develop new therapeutic strategies for these patients, our laboratory has developed a preclinical testing pipeline. Given that histone deacetylase (HDAC) inhibition has been proposed for HB, we hypothesized that we could find an effective combination treatment strategy utilizing HDAC inhibition. METHODS: RNA sequencing, microarray, NanoString, and immunohistochemistry data of patient HB samples were analyzed for HDAC class expression. Patient-derived spheroids (PDSp) were used to screen combination chemotherapy with an HDAC inhibitor, panobinostat. Patient-derived xenograft (PDX) mouse models were developed and treated with the combination therapy that showed the highest efficacy in the PDSp drug screen. RESULTS: HDAC RNA and protein expression were elevated in HB tumors compared to normal livers. Panobinostat (IC50 of 0.013-0.059 µM) showed strong in vitro effects and was associated with lower cell viability than other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). All four models responded to VIP therapy with a decrease in tumor size compared to placebo. After 6 weeks of treatment, two models demonstrated necrotic cell death, with lower Ki67 expression, decreased serum alpha fetoprotein and reduced tumor burden compared to paired VI- and placebo-treated groups. CONCLUSIONS: Utilizing a preclinical HB pipeline, we demonstrate that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from patients with high-risk, relapsed, and treatment-refractory HB. IMPACT AND IMPLICATIONS: Patients with treatment-refractory hepatoblastoma have limited treatment options with survival rates of less than 50%. Our manuscript demonstrates that combination therapy with vincristine, irinotecan, and panobinostat reduces the size of high-risk, relapsed, and treatment-refractory tumors. With this work we provide preclinical evidence to support utilizing this combination therapy as an arm in future clinical trials.

Development of Iodinated Indocyanine Green Analogs as a Strategy for Targeted Therapy of Liver Cancer.

Liver cancer is one of the leading causes of cancer-related deaths, with a significant increase in incidence worldwide. Novel therapies are needed to address this unmet clinical need. Indocyanine green (ICG) is a broadly used fluorescence-guided surgery (FGS) agent for liver tumor resection and has significant potential for conversion to a targeted therapy. Here, we report the design, synthesis, and investigation of a series of iodinated ICG analogs (I-ICG), which can be used to develop ICG-based targeted radiopharmaceutical therapy. We applied a CRISPR-based screen to identify the solute carrier transporter, OATP1B3, as a likely mechanism for ICG uptake. Our lead I-ICG compound specifically localizes to tumors in mice bearing liver cancer xenografts. This study introduces the chemistry needed to incorporate iodine onto the ICG scaffold and defines the impact of these modifications on key properties, including targeting liver cancer in vitro and in vivo.

Pleurisy secondary to nonthrombotic pulmonary emboli in a patient with intravenous drug use.

Patients with injection drug use can have nonthrombotic pulmonary emboli (NTPE) of foreign insoluble particles that are either parts of the equipment used or mixed with the drug as an additive. These foreign particles can become a nidus for infection and inflammation. We present a case of a 31-year-old man with active intravenous drug use who initially presented with signs and symptoms of pleurisy and was found to have NTPE of septic refractile crystalline material as seen on bronchial wash and brush biopsy. We believe our patient likely had embolism of either crack particles, needle fragments or cotton-wool fragments that led to a localized inflammatory reaction and infection. This highlights the importance of obtaining detailed history and diagnostic workup. Once the diagnoses of bacterial endocarditis and thrombophlebitis are ruled out with blood cultures, transthoracic echocardiogram, trans-esophageal echocardiogram and/or CT scan (depending on the suspicion), NTPE should be considered and bronchoscopy with bronchoalveolar lavage with biopsy should be performed.

An indocyanine green-based liquid biopsy test for circulating tumor cells for pediatric liver cancer.

Background and Aims: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the most common malignant hepatocellular tumors seen in children. The aim of this work was to develop a liquid biopsy test for circulating tumor cells (CTCs) for these tumors that would be less invasive and provide information about the real-time state of tumors in response to therapies. Methods: For this test, we utilized indocyanine green (ICG), a far-red fluorescent dye that is used clinically to identify malignant liver cells in the body during surgery. We assessed ICG accumulation in cell lines with fluorescence microscopy and flow cytometry. For our CTC test, we developed a panel of liver tumor-specific markers, ICG, Glypican-3 (GPC3), and DAPI and tested this panel with cell lines and non-cancer control blood samples. We then used this panel to analyze whole blood samples for CTC burden with a cohort of 14 HB and HCC patients and correlated with patient characteristics and outcomes. Results: We showed that ICG accumulation is specific to liver cancer cells, compared to non-malignant liver cells, non-liver solid tumor cells, and non-malignant cells and can be used to identify liver tumor cells in a mixed population of cells. Experiments with the ICG/GPC3/DAPI panel showed that it specifically tagged malignant liver cells. With patient samples, we found that CTC burden from sequential blood samples from the same patients mirrored the patients' responses to therapy. Conclusions: Our novel ICG-based liquid biopsy test for CTCs can be used to specifically count CTCs in the blood of pediatric liver cancer patients. Impact and implications: This manuscript represents the first report of circulating tumor cells in the blood of pediatric liver cancer patients. The novel and innovative assay for CTCs shown in this paper will facilitate future work examining the relationship between CTC numbers and patient outcomes, forming the foundation for incorporation of liquid biopsy into routine clinical care for these patients. Graphical abstract: Overview of novel liquid biopsy test for circulating tumor cells for pediatric liver cancer. Figure made with Biorender.

Repurposing Pitavastatin and L-Glutamine: Replenishing ß-Cells in Hyperlipidemic Type 2 Diabetes Mouse Model.

Type 2 diabetes (T2D) is associated with obesity and declining ß-cells. L-glutamine has been implicated in the amelioration of T2D by virtue of its incretin secretagogue property while, there are mixed reports on pitavastatin's adiponectin potentiating ability. We aimed to investigate the effect of pitavastatin (P), L-glutamine (LG), and combination (P + LG) on glycemic control and ß-cell regeneration in a high-fat diet (HFD) + streptozotocin (STZ)-induced T2D mouse model. C57BL6/J mice treated with HFD + STZ were divided into four groups: diabetes control (HFD + STZ), P, LG, and P + LG, while the control group (NCD) was fed with the normal-chow diet. Significant amelioration was observed in the combination therapy as compared to monotherapies in respect of (i) insulin resistance, glucose intolerance, lipid profile, adiponectin levels, and mitochondrial complexes I, II, and III activities, (ii) reduced phosphoenolpyruvate carboxykinase, glucose 6-phophatase, glycogen phosphorylase, and GLUT2 transcript levels with increased glycogen content in the liver, (iii) restoration of insulin receptor 1ß, pAkt/Akt, and AdipoR1 protein levels in skeletal muscle, and (iv) significant increase in islet number due to ß-cell regeneration and reduced ß-cell death. L-glutamine and pitavastatin in combination can ameliorate T2D by inducing ß-cell regeneration and regulating glucose homeostasis.

Code Status Orders: Do the Options Matter?

BACKGROUND: Code status orders in hospitalized patients guide urgent medical decisions. Inconsistent terminology and treatment options contribute to varied interpretations. OBJECTIVE: To compare two code status order options, traditional (three option) and modified to include additional care options (four option). DESIGN: Prospective, randomized, cross-sectional survey conducted on February-March 2020. Participants were provided with six clinical scenarios and randomly assigned to the three or four option code status order. In three scenarios, participants determined the most appropriate code status. Three scenarios provided clinical details and code status and respondents were asked whether they would provide a particular intervention. This study was conducted at three urban, academic hospitals. PARTICIPANTS: Clinicians who routinely utilize code status orders. Of 4006 participants eligible, 549 (14%) were included. MAIN MEASURES: The primary objective was consensus (most commonly selected answer) based on provided code status options. Secondary objectives included variables associated with participant responses, participant code status model preference, and participant confidence about whether their selections would match their peers. KEY RESULTS: In the three scenarios participants selected the appropriate code status, there was no difference in consensus for the control scenario, and higher consensus in the three option group (p-values < 0.05) for the remaining two scenarios. In the scenarios to determine if a clinical intervention was appropriate, two of the scenarios had higher consensus in the three option group (p-values 0.018 and < 0.05) and one had higher consensus in the four option group (p-value 0.001). Participants in the three option model were more confident that their peers selected the same code status (p-value 0.0014); however, most participants (72%) preferred the four option model. CONCLUSIONS: Neither code status model led to consistent results. The three option model provided consistency more often; however, the majority of participants preferred the four option model.

Contained Left Ventricular Free Wall Rupture Following a Silent Myocardial Infarction.

A left ventricular pseudoaneurysm (LVP) is defined as an outpouching contained by the surrounding pericardium. Clinical presentation is often unspecific with patients presenting with chest pain, dyspnea, symptoms consistent with heart failure, and post-myocardial infarction. Cardiac magnetic resonance imaging represents an important tool for differentiating a pseudoaneurysm from a true aneurysm. Furthermore, multiple imagining modalities are available, including transesophageal and transthoracic echocardiogram and contrast ventriculography, which remains the gold standard diagnostic technique. Early recognition and prompt surgical management are of utmost importance in patients with acute and symptomatic LVP. On the other hand, medical management may be considered in patients with chronic and small pseudoaneurysms. Here, we are presenting a 74-year-old lady who presented with chest pain and was found to have a chronic and small LVP which was managed conservatively.

Hierarchical planning with state abstractions for temporal task specifications.

We often specify tasks for a robot using temporal language that can include different levels of abstraction. For example, the command "go to the kitchen before going to the second floor" contains spatial abstraction, given that "floor" consists of individual rooms that can also be referred to in isolation ("kitchen", for example). There is also a temporal ordering of events, defined by the word "before". Previous works have used syntactically co-safe Linear Temporal Logic (sc-LTL) to interpret temporal language (such as "before"), and Abstract Markov Decision Processes (AMDPs) to interpret hierarchical abstractions (such as "kitchen" and "second floor"), separately. To handle both types of commands at once, we introduce the Abstract Product Markov Decision Process (AP-MDP), a novel approach capable of representing non-Markovian reward functions at different levels of abstractions. The AP-MDP framework translates LTL into its corresponding automata, creates a product Markov Decision Process (MDP) of the LTL specification and the environment MDP, and decomposes the problem into subproblems to enable efficient planning with abstractions. AP-MDP performs faster than a non-hierarchical method of solving LTL problems in over 95 % of path planning tasks, and this number only increases as the size of the environment domain increases. In a cleanup world domain, AP-MDP performs faster in over 98 % of tasks. We also present a neural sequence-to-sequence model trained to translate language commands into LTL expression, and a new corpus of non-Markovian language commands spanning different levels of abstraction. We test our framework with the collected language commands on two drones, demonstrating that our approach enables robots to efficiently solve temporal commands at different levels of abstraction in both indoor and outdoor environments.

Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy.

Disruption of circadian glucocorticoid oscillations in Cushing's disease and chronic stress results in obesity and adipocyte hypertrophy, which is believed to be a main source of the harmful effects of obesity. Here, we recapitulate stress due to jet lag or work-life imbalances by flattening glucocorticoid oscillations in mice. Within 3 days, mice achieve a metabolic state with persistently high insulin, but surprisingly low glucose and fatty acids in the bloodstream, that precedes a more than 2-fold increase in brown and white adipose tissue mass within 3 weeks. Transcriptomic and Cd36-knockout mouse analyses show that hyperinsulinemia-mediated de novo fatty acid synthesis and Cd36-mediated fatty acid uptake drive fat mass increases. Intriguingly, this mechanism by which glucocorticoid flattening causes acute hyperinsulinemia and adipocyte hypertrophy is unexpectedly beneficial in preventing high levels of circulating fatty acids and glucose for weeks, thus serving as a protective response to preserve metabolic health during chronic stress.

HepT1-derived murine models of high-risk hepatoblastoma display vascular invasion, metastasis, and circulating tumor cells.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.

A novel therapeutic combination of sitagliptin and melatonin regenerates pancreatic ß-cells in mouse and human islets.

Autoimmune-led challenge resulting in ß-cell loss is responsible for the development of type 1 diabetes (T1D). Melatonin, a pineal hormone or sitagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor, has increased ß-cell mass in various diabetic models and has immunoregulatory property. Both ß-cell regenerative capacity and melatonin secretion decrease with ageing. Thus, we aimed to investigate the therapeutic potential of melatonin combined with sitagliptin on ß-cell regeneration under glucotoxic stress, in the streptozotocin-induced young and old diabetic mouse models, and euglycemic humanized islet transplant mouse model. Our results suggest that combination therapy of sitagliptin and melatonin show an additive effect in inducing mouse ß-cell regeneration under glucotoxic stress, and in the human islet transplant mouse model. Further, in the young diabetic mouse model, the monotherapies induce ß-cell transdifferentiation and reduce ß-cell apoptosis whereas, in the old diabetic mouse model, melatonin and sitagliptin induce ß-cell proliferation and ß-cell transdifferentiation, and it also reduces ß-cell apoptosis. Further, in both the models, combination therapy reduces fasting blood glucose levels, increases plasma insulin levels and glucose tolerance and promotes ß-cell proliferation, ß-cell transdifferentiation, and reduces ß-cell apoptosis. It can be concluded that combination therapy is superior to monotherapies in ameliorating diabetic manifestations, and it can be used as a future therapy for ß-cell regeneration in diabetes patients.

Calorie restriction potentiates the therapeutic potential of GABA in managing type 2 diabetes in a mouse model.

Dysfunctional adipocytes/ß-cells advance type 2 diabetes (T2D). Calorie restriction (CR) improves insulin sensitivity and fasting blood glucose (FBG) levels, while γ-aminobutyric acid (GABA) exerts regenerative effects. The impact of therapies was assessed by a high-fat diet (HFD) + streptozotocin (STZ) induced T2D mouse model. The mice were fed a CR diet (30% reduction of HFD) and treated with GABA (2.5 mg/kg i.p) for 5 weeks. Standard protocols were used to assess metabolic parameters. The mRNA expression was monitored by SYBR Green-qPCR in the targeted tissues. Oxygen consumption rate in the mitochondrial complexes was evaluated by oxytherm clark-type oxygen electrode. Pancreatic ß-cell regeneration and apoptosis were analysed by immunohistochemistry. CR + GABA combination therapy showed improved metabolic parameters compared to the monotherapies. We have observed improved transcript levels of G6Pase, PEPCK, Glycogen Phosphorylase, GLUT2 and GCK in liver; ACC and ATGL in adipose tissue. Also increased SIRT-1, PGC-1α and TFAM expression; up-regulated mitochondrial complexes I-III activities were observed. We have seen increased BrdU/Insulin and PDX1/Ngn3/Insulin co-positive cells in CR + GABA treated group with a reduction in apoptotic marker (TUNEL/Insulin co-positive cells). Our results indicate that CR in combination with GABA ameliorates T2D in HFD + STZ treated mice by GABA induced ß-cell regeneration, and CR mediated insulin sensitivity.

Diabetes mellitus and melatonin: Where are we?

Diabetes mellitus (DM) and diabetes-related complications are amongst the leading causes of mortality worldwide. The international diabetes federation (IDF) has estimated 592 million people to suffer from DM by 2035. Hence, finding a novel biomolecule that can effectively aid diabetes management is vital, as other existing drugs have numerous side effects. Melatonin, a pineal hormone having antioxidative and anti-inflammatory properties, has been implicated in circadian dysrhythmia-linked DM. Reduced levels of melatonin and a functional link between melatonin and insulin are implicated in the pathogenesis of type 2 diabetes (T2D). Additionally, genomic studies revealed that rare variants in melatonin receptor 1b (MTNR1B) are also associated with impaired glucose tolerance and increased risk of T2D. Moreover, exogenous melatonin treatment in cell lines, rodent models, and diabetic patients has shown a potent effect in alleviating diabetes and other related complications. This highlights the role of melatonin in glucose homeostasis. However, there are also contradictory reports on the effects of melatonin supplementation. Thus, it is essential to explore if melatonin can be taken from bench to bedside for diabetes management. This review summarizes the therapeutic potential of melatonin in various diabetic models and whether it can be considered a safe drug for managing diabetic complications and diabetic manifestations like oxidative stress, inflammation, ER stress, mitochondrial dysfunction, metabolic dysregulation, etc.

Crucial Conversations for High-Risk Populations before Surgery: Advance Care Planning in a Preoperative Setting.

Background: High-risk patients undergoing elective surgery are at risk for perioperative complications, including readmissions and death. Advance care planning (ACP) may allow for preparation for such events. Objectives: (1) To assess the completion rate of advance directives (ADs) and their association with one year readmissions and mortality (2) to examine clinical events for decedents. Design: This is an observational cohort study conducted through chart review. Setting/Subjects: Subjects were 400 patients undergoing preoperative evaluation for elective surgery at two hospitals in the United States. Measurements: The prevalence of ADs at the time of surgery and at one year, readmissions, and mortality at one year were determined. Results: Three-hundred ninety patients were included. In total, 102 (26.4%) patients were readmitted, yet did not complete an AD. Seventeen (4.4%) patients filed an AD during follow-up. Nineteen patients died and mortality rate was 4.9%. There was a significant association between completing an AD before death. Of the decedents, seven (37%) underwent resuscitation, but only four had ADs. Conclusions: Many high-risk surgical patients would benefit from ADs before clinical decline. Preoperative clinics present a missed opportunity to ensure ACP occurs before complications arise.

Partnership development of the COVID-19 Front Door: a best evidence resource.

This project describes the creation of a single searchable resource during the pandemic, called the COVID-19 Best Evidence Front Door, with a primary goal of providing direct access to high-quality meta-analyses, literature syntheses, and clinical guidelines from a variety of trusted sources. The Front Door makes relevant evidence findable and accessible with a single search to aggregated evidence-based resources, optimizing time, discovery, and improved access to quality scientific evidence while reducing the burden of frontline health care providers and other knowledge-seekers in needing to separately identify, locate, and explore multiple websites.