Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.

T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.

Intratumoral Cell Neighborhoods Coordinate Outcomes in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease characterized by a spatially heterogeneous tumor microenvironment. Within the PDA microenvironment, cells organize into communities where cell fate is influenced by neighboring cells of diverse ontogeny and function. However, it remains unclear how cell neighborhoods in the tumor microenvironment evolve with treatment and impact clinical outcomes. METHODS: Here, using automated chromogenic multiplex immunohistochemistry and unsupervised computational image analysis of human PDA tumors, we investigated cell neighborhoods in surgically resected tumors from patients with chemotherapy-naïve PDA (n = 59) and neoadjuvant chemotherapy-treated PDA (n = 57). Single cells were defined by lineage markers (CD3, CD8, Foxp3, CD68, CK19), proliferation (Ki67), and neighboring cells. RESULTS: Distinct intratumoral immune and tumor cell subsets were defined by neighboring cells. Higher content of stromal-associated macrophages was seen in chemotherapy-naïve tumors from long-term survivors (overall survival >3 years) compared with short-term survivors (overall survival <1 year), whereas immune-excluded tumor cells were higher in short-term survivors. Chemotherapy-treated vs -naïve tumors showed lower content of tumor-associated T cells and macrophages but similar densities of stromal-associated immune cells. However, proliferating tumor cell subsets with immune-rich neighborhoods were higher in chemotherapy-treated tumors. In a blinded analysis of tumors from patients treated with neoadjuvant chemotherapy, a composite index comprising lower quantities of immune-excluded tumor cells and higher spatially distinct immune cell subsets was associated with prolonged survival. CONCLUSIONS: Together, these data provide new insights into discrete cell communities in PDA and show their clinical relevance.

Recurrent and functional regulatory mutations in breast cancer.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.

Stimuli-responsive transdermal microneedle patches.

Microneedle (MN) patches consisting of miniature needles have emerged as a promising tool to perforate the stratum corneum and translocate biomolecules into the dermis in a minimally invasive manner. Stimuli-responsive MN patches represent emerging drug delivery systems that release cargos on-demand as a response to internal or external triggers. In this review, a variety of stimuli-responsive MN patches for controlled drug release are introduced, covering the mechanisms of action toward different indications. Future opportunities and challenges with respect to clinical translation are also discussed.

Infants with pulmonary atresia intact ventricular septum who require balloon atrial septostomy have significantly higher 18-month mortality.

INTRODUCTION: Many newborns with pulmonary atresia/intact ventricular septum require intervention to establish pulmonary flow and sufficient cardiac output. The resulting haemodynamic changes are not well characterised and may have unintended consequences. METHODS: This is a 30-year (1988-2018) retrospective study of patients with pulmonary atresia intact ventricular septum. RESULTS: Eighty-nine patients were included, and median follow-up was 8 years. Fifty-five per cent had coronary sinusoids and 27% had right ventricular-dependent coronary circulation. Most patients were managed with surgical aortopulmonary or modified Blalock-Taussig shunt (73%), and 12 patients underwent balloon atrial septostomy before surgical intervention. The remaining patients (27%) underwent only transcatheter interventions; 7 required an atrial septostomy and 17 required ductal stentings. All-cause mortality was 10%, most deaths (89%) occurred before 18 months of age. Of these early deaths, 87% required a balloon atrial septostomy and 85% had right ventricular-dependent coronary sinusoids. Eighteen-month mortality was significantly higher for patients who required a balloon atrial septostomy compared to those who did not (36% versus 1.4% p < 0.0001). DISCUSSION: Patients with pulmonary atresia/intact ventricular septum who require balloon atrial septostomy in the newborn period have significantly higher 18-month mortality. Quantifying the mortality difference may help guide prognostication and expectation setting. Infants who had septostomy and a surgical shunt in the newborn period fared better than those who only underwent septostomy (even when accompanied by ductal stenting). For infants with right ventricular-dependent circulation, atrial septostomy should only be performed on an urgent or emergent basis and these patients should be considered for early surgical intervention and neonatal transplant.

Point-of-Care Ultrasound and the Septic Prosthetic Hip Joint.

Septic joints can have an insidious onset and are difficult to diagnosis. Diagnosis can be more complicated in the setting of a distant prosthetic joint. Plain films and inflammatory markers are not specific and magnetic resonance imaging (MRI) is not a timely test in the emergency department. Computed tomography (CT) scan is quick and useful to evaluate for bony changes or signs of inflammation, but lacks the details of MRI, and the prosthetic joint may cause significant artifact. Point-of-care ultrasound (POCUS) is often used in the pediatric population to evaluate for an effusion when there is a concern for a septic native hip joint and is finding a role in adult emergency medicine to evaluate for an effusion in painful native adult hip joints. Even so, ultrasound is not currently included in diagnostic algorithms for diagnosing prosthetic hip joint infections (PJIs). POCUS is, however, readily available in the emergency department. We present a case where POCUS aided in identifying a periprosthetic synovitis and changed the course of the patient's management from previous physical therapy to an investigation toward the final diagnosis of a septic prosthetic hip joint.

Non-anastomotic failure of woven Dacron tube grafts in the thoracic aorta in young adults.

BACKGROUND: Dacron tube grafts have been used in the surgical management of cardiovascular disease since the 1970s. Complications at the site of the anastomosis have been well described. Non-anastomotic failure is far less common. We present a series of four patients who presented with complications of non-anastomotic failure of woven Dacron tube grafts. METHODS: A retrospective chart review of four patients who presented to our institution between March 2014 and March 2017 with clinical complications of a Dacron tube graft was conducted. RESULTS: All four patients underwent a staged surgical repair for an interrupted aortic arch between the years of 1988 and 2001. All four patients underwent revision of their original interposition graft (Gore-Tex, W.L. Gore & Associates, Flagstaff, AZ) with implantation of a Hemashield woven Dacron tube graft (Maquet, Rastatt, Germany). From 13 to 22 years postimplant of the Dacron tube graft, all patients presented with symptoms or clinical evidence of primary graft failure. Two patients underwent urgent surgical intervention and did not survive. One patient underwent attempted surgical intervention, which was aborted in the setting of profuse bleeding, and ultimately had an endovascular rescue of the tube graft with a Zenith Alpha endograft (Cook Medical, Bloomington, IN). One patient underwent elective endovascular intervention prior to onset of symptoms. CONCLUSION: Non-anastomotic failure of woven Dacron tube grafts can occur in the thoracic aorta in young adults and may be managed with endovascular techniques.

Descriptive Data of Transgender and Nonbinary People's Experiences of Sexual Assault: Context, Perpetrator Characteristics, and Reporting Behaviors.

Purpose: Nearly half of transgender and nonbinary (trans/NB) people will experience sexual assault in their lifetime. Beyond prevalence, little else is known about the general context in which sexual assault occurs in this community. In addition, whether and to whom trans/NB people report these experiences is also not well understood. As such, we examined these contextual factors regarding trans/NB people's sexual assault experiences. Methods: Using a cross-sectional online survey, we assessed sexual assault prevalence rates, contextual details, and reporting behaviors in a sample of 230 trans/NB adults in the United States. Data were gathered in 2016 and 2017 across two collections. Results: Eighty percent of the sample had experienced sexual assault; 62% reported experiencing completed penetrative behaviors (rape) and 37% reported repeat victimization-assault during both childhood and adulthood. Most participants indicated that their perpetrator was male (80%) and the plurality described perpetrators as dating partners (34%) or acquaintances (34%). Alcohol was involved in 26% of assaults. Approximately 8% characterized the event as a hate crime. Most trans/NB people reported their experience to a friend (64%), dating partner (50%), and/or therapist (35%). Only 6% reported their sexual assault to the police, and 20% stated that they have never told anyone. Conclusion: With the exception of substantially higher prevalence rates, many of our findings are similar to findings in the broader, typically cis-centric, sexual assault literature. We recommend that prevention and support services address both the high rates of sexual assault and the low rates of reporting to police and other support services.

Osteoinductive Dental Pulp Stem Cell-Derived Extracellular Vesicle-Loaded Multifunctional Hydrogel for Bone Regeneration.

Stem cell-derived extracellular vesicles (EVs) show great potential for promoting bone tissue regeneration. However, normal EVs (Nor-EVs) have a limited ability to direct tissue-specific regeneration. Therefore, it is necessary to optimize the osteogenic capacity of EV-based systems for repairing extensive bone defects. Herein, we show that hydrogels loaded with osteoinductive dental pulp stem cell-derived EVs (Ost-EVs) enhanced bone tissue remodeling, resulting in a 2.23 ± 0.25-fold increase in the expression of bone morphogenetic protein 2 (BMP2) compared to the hydrogel control group. Moreover, Ost-EVs led to a higher expression of alkaline phosphatase (ALP) (1.88 ± 0.16 of Ost-EVs relative to Nor-EVs) and the formation of orange-red calcium nodules (1.38 ± 0.10 of Ost-EVs relative to Nor-EVs) in vitro. RNA sequencing revealed that Ost-EVs showed significantly high miR-1246 expression. An ideal hydrogel implant should also adhere to surrounding moist tissues. In this study, we were drawn to mussel-inspired adhesive modification, where the hydrogel carrier was crafted from hyaluronic acid (HA) and polyethylene glycol derivatives, showcasing impressive tissue adhesion, self-healing capabilities, and the ability to promote bone growth. The modified HA (mHA) hydrogel was also responsive to environmental stimuli, making it an effective carrier for delivering EVs. In an ectopic osteogenesis animal model, the Ost-EV/hydrogel system effectively alleviated inflammation, accelerated revascularization, and promoted tissue mineralization. We further used a rat femoral condyle defect model to evaluate the in situ osteogenic ability of the Ost-EVs/hydrogel system. Collectively, our results suggest that Ost-EVs combined with biomaterial-based hydrogels hold promising potential for treating bone defects.

Serum Amyloid A Proteins and Their Impact on Metastasis and Immune Biology in Cancer.

Cancer triggers the systemic release of inflammatory molecules that support cancer cell metastasis and immune evasion. Notably, this biology shows striking similarity to an acute phase response that is coordinated by the liver. Consistent with this, a role for the liver in defining cancer biology is becoming increasingly appreciated. Understanding the mechanisms that link acute phase biology to metastasis and immune evasion in cancer may reveal vulnerable pathways and novel therapeutic targets. Herein, we discuss a link between acute phase biology and cancer with a focus on serum amyloid A proteins and their involvement in regulating the metastatic cascade and cancer immunobiology.

Patent Ductus Arteriosus Stenting for All Ductal-Dependent Cyanotic Infants: Waning Use of Blalock-Taussig Shunts.

[Figure: see text].

Transjugular liver biopsy for Fontan associated liver disease surveillance: Technique, outcomes and hemodynamic correlation.

BACKGROUND: Fontan associated liver disease (FALD) is attributed to the limitations of the Fontan circulation, resulting in congestive hepatopathy. The technique and outcomes of transjugular liver biopsies (TJLB) in Fontan patients warrant definition as part of a rigorous FALD surveillance program. METHOD: Four year review of patients with Fontan physiology who underwent a TJLB during surveillance catheterizations. Biopsy site, technique, histology, angiography, hemodynamics, and complications were reviewed to assess correlation of biopsy scores with simultaneously obtained catheterization hemodynamics. RESULTS: 125 patients with a TJLB from 10/1/14 to 5/1/18. Median age 17 years (2-50.5). Technical success 100% (125/125), all samples diagnostic. 17% (21) accessed via the left internal jugular vein, secondary to right IJ occlusion or Heterotaxy syndrome. No patients had superior compartment obstruction preventing transjugular approach. 3.2% complication rate (4/125). Complications were early in the experience, including capsular perforation (2), renal hematoma (1) and hemobilia (1), all without long-term effect and all avoidable. After standardized entry/exit angiography was adopted, no further complications noted. There is a significant correlation between the newly described modified Ishak congestive hepatic fibrosis (ICHF) score with mean Fontan pressure, time from Fontan and cardiac index. CONCLUSIONS: TJLB is an alternate method for obtaining critical FALD surveillance data, with lower complication rates that traditional techniques. Vascular anomalies in Fontan physiology appear common and warrant pre-biopsy assessment. There is a significant correlation between biopsy score, time from Fontan, mean Fontan pressure and cardiac index.

TNF blockade uncouples toxicity from antitumor efficacy induced with CD40 chemoimmunotherapy.

Agonist CD40 antibodies are under clinical development in combination with chemotherapy as an approach to prime for antitumor T cell immunity. However, treatment with anti-CD40 is commonly accompanied by both systemic cytokine release and liver transaminase elevations, which together account for the most common dose-limiting toxicities. Moreover, anti-CD40 treatment increases the potential for chemotherapy-induced hepatotoxicity. Here, we report a mechanistic link between cytokine release and hepatotoxicity induced by anti-CD40 when combined with chemotherapy and show that toxicity can be suppressed without impairing therapeutic efficacy. We demonstrate in mice and humans that anti-CD40 triggers transient hepatotoxicity marked by increased serum transaminase levels. In doing so, anti-CD40 sensitizes the liver to drug-induced toxicity. Unexpectedly, this biology is not blocked by the depletion of multiple myeloid cell subsets, including macrophages, inflammatory monocytes, and granulocytes. Transcriptional profiling of the liver after anti-CD40 revealed activation of multiple cytokine pathways including TNF and IL-6. Neutralization of TNF, but not IL-6, prevented sensitization of the liver to hepatotoxicity induced with anti-CD40 in combination with chemotherapy without impacting antitumor efficacy. Our findings reveal a clinically feasible approach to mitigate toxicity without impairing efficacy in the use of agonist CD40 antibodies for cancer immunotherapy.

Paracrine and cell autonomous signalling in pancreatic cancer progression and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) shows remarkable propensity to metastasize. This predilection to escape from the primary tumor is driven by paracrine and autocrine mechanisms that guide cancer cells through a multi-step process concluding with colonization in distant tissues. Although cell-intrinsic features support the metastatic ability of cancer cells, permissive microenvironments within the primary organ and at sites of distant metastasis may be rate-limiting. Identification of cancer cell-extrinsic factors that regulate formation of these environments lend new therapeutic targets for intervening on the metastatic cascade. In addition, the bipolar, yet fundamental, role of the immune system in the metastatic process presents therapeutic opportunities. Herein, we review the current knowledge of the metastatic cascade in PDAC, and propose that genomically stable determinants of metastasis (e.g. the pro-metastatic niche and immune system) are actionable targets for preventing, containing, and treating metastasis in PDAC.

Stenting the vertical neonatal ductus arteriosus via the percutaneous axillary approach.

BACKGROUND/OBJECTIVE: Stenting the ductus arteriosus (DAS) has become an alternative to surgical systemic to pulmonary artery shunts in neonates with ductal-dependent pulmonary blood flow (PBF). Femoral approach for a vertical ductus can be difficult secondary to the acute angle and tortuous course, thus alternative access sites have been explored. Carotid access complications have been reported in 5%-10%. The extensive use of an axillary arterial approach in the United States has not been reported. The aim of this study is to describe our experience with DAS using the axillary approach. METHODS: We reviewed all patients with DAS with an axillary approach in neonates with ductal-dependent PBF (May 2017-May 2018) in our institution. Procedural reports, angiograms, and clinical records of all consecutive patients were reviewed. Procedural technique, procedural outcomes, adverse events, and post-hospital courses are reported. RESULTS: Seven consecutive patients who received DAS utilizing axillary approach. All patients had ductal-dependent PBF through a vertical, tortuous ductus. Five had pulmonary atresia or near atresia, one had compromised PBF due to dynamic subvalvar obstruction, and one had Tetralogy of Fallot with isolated left pulmonary artery. Axillary access with 3.3 or 4 French sheath was obtained using ultrasound guidance. Bare metal coronary stents were deployed successfully in all. Intra-procedure, one developed in stent thrombus requiring re-stenting. There were no procedural mortalities or major adverse events from axillary access. There is a steep learning curve. Hemostasis was achieved with manual compression. Two patients had reintervention at 6-8 weeks. All patients underwent successful planned surgeries. CONCLUSIONS: This series suggests DAS in neonates utilizing an axillary approach is a feasible and effective alternative for establishing PBF. Axillary arterial approach may be preferred as there is no risk to neurological sequelae and very low risk of limb complications. Larger series are needed to validate this approach.

Pulmonary artery interventions after the arterial switch operation: Unique and significant risks.

BACKGROUND: In the modern era, results of the arterial switch operation (ASO) for transposition of the great arteries are excellent. However, because of the LeCompte maneuver, there may be a propensity for development of pulmonary artery stenosis. We encountered atypical complications of pulmonary artery stenting in patients after the ASO, including aorto-pulmonary fistula and coronary compression. METHODS: We performed a 10-year retrospective review of catheterizations performed in patients after ASO in our institution with a focus on adverse events. RESULTS: Diagnostic and interventional catheterizations were performed in 47 patients. In 29 patients, 37 interventional procedures performed, which included pulmonary artery angioplasty and/or stenting. In this group, there were five major adverse events (14%), including three aorto-pulmonary fistulae and one coronary artery compression among patients having stent implantation or stent redilation. In addition, there were 6/37 (16%) intended stent procedures, which were aborted because there appeared to be high-risk of significant adverse events. CONCLUSIONS: This review suggests that percutaneous intervention on pulmonary artery stenosis after ASO has high-risk and should be undertaken advisedly. Prior thorough evaluation of coronary arteries is mandatory as coronary reimplantation sites may be adjacent to sites of pulmonary artery stenosis. Furthermore, if pulmonary artery stent implantation or stent redilation is contemplated, the risk of stent fracture and possible AP fistula should be recognized. Primary use of reinforced covered stents should be considered.

A Nanopore Sequencing-Based Assay for Rapid Detection of Gene Fusions.

Structural chromosomal rearrangements leading to gene fusions are strong driver mutations in a variety of tumors. Identification of specific gene fusions can be essential for distinguishing benign from malignant conditions and for recognizing specific subtypes of neoplasms that can have different management and prognosis. Rapid identification of gene fusions is particularly critical for patients with acute leukemia who cannot wait more than a few days before initiating treatment and for whom treatment can be dramatically different depending on the leukemia subtype. We have developed an assay for rapid detection of oncogenic gene fusions (within 24 hours) that takes advantage of the long reads and real-time data generation of the Oxford Nanopore MinION sequencing system. By using a modification of the anchored multiplex PCR method for library construction, we confidently identified BCR-ABL1 fusion transcripts, with >100 reads within 15 minutes of sequencing. By using formalin-fixed, paraffin-embedded specimens routinely tested in our clinical molecular laboratory, fusions were successfully identified within 5 hours from acquisition of Illumina-ready libraries and 30 minutes of sequencing initiation, including cases diluted to a tumor fraction of 5%. In conclusion, we have developed a nanopore-based sequencing assay that can decrease turnaround time for detection of fusion oncogenes and may be a valid approach for laboratories with low specimen volume and for cases in need of rapid results.

Clinical Validation of a Cell-Free DNA Gene Panel.

The use of liquid biopsies to identify driver mutations in patients with solid tumors holds great promise for performing targeted therapy selection, monitoring disease progression, and detecting treatment resistance mechanisms. We describe herein the development and clinical validation of a 28-gene cell-free DNA panel that targets the most common genetic alterations in solid tumors. Bioinformatic and variant filtering solutions were developed to improve test sensitivity and specificity. The panel and these tools were used to analyze commercially available controls, allowing establishment of a limit of detection allele fraction cutoff of 0.25%, with 100% (95% CI, 81.5%-100%) specificity and 89.8% (95% CI, 81.0%-94.9%) sensitivity. In addition, we analyzed a total of 163 blood samples from patients with metastatic cancer (n = 123) and demonstrated a >90% sensitivity for detecting previously identified expected mutations. Longitudinal monitoring of patients revealed a strong correlation of variant allele frequency changes and clinical outcome. Additional clinically relevant information included identification of resistance mutations in patients receiving targeted treatment and detection of complex patterns of mutational heterogeneity. Achieving lower limits of detection will require additional improvements to molecular barcoding; however, these data strongly support clinical implementation of cell-free DNA panels in advanced cancer patients.

Highly Multiplexed Fluorescence in Situ Hybridization for in Situ Genomics.

The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but it is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent bar-code system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. We demonstrate the reliability of this multiplex approach on normal human lymphocytes, metaphase spreads of transformed cell lines, and cultured circulating tumor cells. It also opens the door to the development of gene panels for more comprehensive analysis of copy number changes in tissue, including the study of heterogeneity and of high-throughput clinical assays that could provide rapid quantification of gene copy numbers in samples with limited cellularity, such as circulating tumor cells.

Evaluation of Fontan liver disease: Correlation of transjugular liver biopsy with magnetic resonance and hemodynamics.

INTRODUCTION: Liver fibrosis and cirrhosis are late complications in Fontan palliation. Liver biopsy is the gold standard. The goal of this study is to correlate transjugular liver biopsy (TJLB) in the setting of Fontan palliation with noninvasive testing and hemodynamics. METHODS: Between August 2014 and July 2017, 49 Fontan patients underwent TJLB. All the patients had hemodynamic evaluation, 28 patients had MRE (magnetic resonance elastography) and 40 patients had cardiopulmonary exercise test. Histologic liver fibrosis was quantitated using traditional histologic scoring systems and a modified Ishak congestive hepatic fibrosis score. RESULTS: Median age 17.8 years, median time since Fontan 15.2 years. Primary diagnosis and Fontan type were variables, but predominantly LV morphology (30/49), lateral tunnel Fontan (29/49), originally fenestrated (37/49), and 11/49 had a pacemaker. Histologic fibrosis correlated with MRE (R = 0.62, P ≤ .001). Histologic fibrosis and MRE correlated with Fontan pressure (R = 0.38, P = .008 & R = 0.59, P ≤ .001). Morphology of the single ventricle did not correlate with liver fibrosis. The presence of a fenestration resulted in a higher cardiac index (P = .026) but did not resulted in lower liver fibrosis (P = .64). CONCLUSION: Noninvasive tests, such as MRE, may be suitable for longitudinal follow-up in patients with single ventricle physiology. Our data suggest that there is reasonable correlation of MRE liver stiffness with biopsy scoring systems and Fontan pressures. We demonstrated the feasibility of TJLB in the setting of Fontan palliation and demonstrated its correlation with noninvasive measures particularly MRE. We recommend selective use of TJLB when MRE score is >5 KPa or when there are other clinical signs of cirrhosis.