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SHP2 inhibitors (SHP2i) alone and in various combinations are being tested in multiple tumors with overactivation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages, induced tumor-intrinsic CCL5/CXCL10 secretion, and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non–small cell lung cancer (NSCLC). However, treatment also increased intratumor granulocytic myeloid-derived suppressor cells (gMDSC) via tumor-intrinsic, NFκB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C inhibitor trials. Combined SHP2 (SHP099)/CXCR1/2 (SX682) inhibition depleted a specific cluster of S100a8/9hi gMDSCs, generated Klrg1+ CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras- and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in patients with NSCLC.Significance:Our study shows that inhibiting the SHP2/RAS/ERK pathway triggers NFκB-dependent upregulation of CXCR2 ligands and recruitment of S100A8hi gMDSCs, which suppress T cells. Combining SHP2/CXCR2 inhibitors blocks gMDSC immigration, resulting in enhanced Th1 polarization, induced CD8+KLRG1+ effector T cells with high cytotoxic activity, and improved survival in multiple NSCLC models.This article is highlighted in the In This Issue feature, p. 1

Table S7

Table S1, S3, S4, S5, S6

Figure S1-6

Table S2

Supplemental Figure S7. SX-682 treatment does not alter PMN-MDSC suppressive capacity

Figure S4. Primary tumor incidence, growth, survival and metastasis in Ptenf/f vs. Arf6Q67L mice. (a) Increased tumor incidence (% = # of mice that developed a tumor / # TVA positive, injected mice) in Ptenf/f vs. Arf6Q67L cohorts. Fisher's exact test, two-tailed, α

Figure S7. Low expression of ARF GAP genes correlates with reduced overall survival. (a) Individual gene plots of four ARF6 GAPs (ACAP1, ADAP1, ARAP2, SMAP2), an ARF1/ARF5 GAP (AGAP2) and one predicted ARF GAP (AGFG2). n = 439 patients. (b) Individual gene plots in Stage III specific cohort, n=142 patients. Log-rank (Mantel-Cox) test.

Figure S6. ARF6-GDP inhibits PI3K-AKT activation and is necessary for AKT activation. (a-b) Quantification of western blots shown in Figure 5c-d. Reproducible reduction in pAKT by Myc-ARF6T27N over expression in A375 cells (n=4), A2058 cells (n=4), and HEY-T30 cells (n=5). Reduced pAKT with siRNA knockdown of ARF6 in SK-MEL-147 (n=5), A2058 (n=4), and CACL (n=4) and HEY-T30 (n=6). Graphs show individual data points normalized to control along with geometric means, 95% CI, ratio paired t test. (c) Phosphokinase array images (left) and quantification of signal by densitometry (right, histogram), n=1. Expression of ARF6T27N in A2058 cells preferentially reduced pAKT and the AKT substrate PRAS40 over most other proteins in the array.

Supplemental Figure S4. Ex vivo SX-682 exposure does not reduce the viability of sorted MDSC

Supplemental Figure S2. PMN-MDSC can be isolated from tumor single cell suspensions

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients.Significance:These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.

Figure S1. Detection of HA-tagged ARF6Q67L in BrafCA;Cdkn2af/f melanoma. a) Anti-HA immunostain of FFPE sections of primary tumors with variable levels of detection of ARF6Q67L -HA. The strongest staining was observed in tumors with the highest level of mRNA detected by qRT-PCR (b). Absent staining of FFPE tumors was observed in tumors with low (6456) and undetectable (6457) levels of mRNA by qRT-PCR of frozen tumors. Scale bars = 20mm, 400x magnification. Control = BrafCA;Cdkn2af/f (Cre-only injection). b) qRT-PCR of Arf6 Q67L-HA from fresh, frozen primary tumor fragments. Bars represent the absolute difference in crossing threshold (Ct) between Arf6Q67L-HA and Gapdh. The Ct threshold that defines detection was established above the background signal for controls in both the BrafCA;Cdkn2af/f and the BrafCA;Cdkn2af/f ;Ptenf/f cohorts (see Figures 1g and S3). With rare exception, positive tumors demonstrated Arf6 Q67L-HA expression levels lower than Gapdh.

Table S1: ARF6 pathways and related genes.

Figure S5: ARF6-GTP induces morphologic changes in mouse melanoma cells. Bright field images (100x magnification) of primary mouse melanoma cell lines derived from control BrafCA;Cdkn2af/f (5588) or BrafCA;Cdkn2af/f + Arf6Q67L (6431, 6455) mice. ARF6-GTP induces elongation/spindling of tumor cells relative to the more polygonal-shaped controls. Scale bars = 400um.

Supplemental Figure S8. SX-682 does not directly alter MOC2 cells in vitro

Purpose:Natural killer (NK)-cell–based immunotherapy may overcome obstacles to effective T-cell–based immunotherapy such as the presence of genomic alterations in IFN response genes and antigen presentation machinery. All immunotherapy approaches may be abrogated by the presence of an immunosuppressive tumor microenvironment present in many solid tumor types, including head and neck squamous cell carcinoma (HNSCC). Here, we studied the role of myeloid-derived suppressor cells (MDSC) in suppressing NK-cell function in HNSCC.Experimental Design:The ability of peripheral and tumor-infiltrating MDSC from mice bearing murine oral cancer 2 (MOC2) non-T-cell–inflamed tumors and from patients with HNSCC to suppress NK-cell function was studied with real-time impedance and ELISpot assays. The therapeutic efficacy of SX-682, a small-molecule inhibitor of CXCR1 and CXCR2, was assessed in combination with adoptively transferred NK cells.Results:Mice bearing MOC2 tumors pathologically accumulate peripheral CXCR2+ neutrophilic-MDSC (PMN-MDSC) that traffic into tumors and suppress NK-cell function through TGFβ and production of H2O2. Inhibition of MDSC trafficking with orally bioavailable SX-682 significantly abrogated tumor MDSC accumulation and enhanced the tumor infiltration, activation, and therapeutic efficacy of adoptively transferred murine NK cells. Patients with HNSCC harbor significant levels of circulating and tumor-infiltrating CXCR1/2+ CD15+ PMN-MDSC and CD14+ monocytic-MDSC. Tumor MDSC exhibited greater immunosuppression than those in circulation. HNSCC tumor MDSC immunosuppression was mediated by multiple, independent, cell-specific mechanisms including TGFβ and nitric oxide.Conclusions:The clinical study of CXCR1/2 inhibitors in combination with adoptively transferred NK cells is warranted.

Supplemental Figure S1. MOC2 tumors express myeloid chemokines

Methods not essential to the main conclusions but important for critical review of methods used for this work

Supplemental Figure S6. SX-682 treatment does not enhance MOC2 tumor T cell infiltration

Figure S3. Arf6Q67L-HA expression in tumors from BrafCA;Cdkn2af/f;Ptenf/f mice. qRT-PCR detection of Arf6 Q67L-HA from fresh, frozen primary tumor fragments. Bars represent the absolute difference in crossing threshold (Ct) between Arf6 Q67L-HA and Gapdh. The Ct threshold that defines detection was established above the background signal for controls in both the BrafCA;Cdkn2af/f and the BrafCA;Cdkn2af/f ;Ptenf/f cohorts (see also Figure S1). All mouse tumor negative by qRT-PCR showed detectable HA-tagged ARF6Q67L by immunohistochemistry (not shown, see Figure S1 for example).

The noninflamed microenvironment in prostate cancer represents a barrier to immunotherapy. Genetic alterations underlying cancer cell–intrinsic oncogenic signaling are increasingly appreciated for their role in shaping the immune landscape. Recently, we identified Pygopus 2 ( PYGO2 ) as the driver oncogene for the amplicon at 1q21.3 in prostate cancer. Here, using transgenic mouse models of metastatic prostate adenocarcinoma, we found that Pygo2 deletion decelerated tumor progression, diminished metastases, and extended survival. Pygo2 loss augmented the activation and infiltration of cytotoxic T lymphocytes (CTLs) and sensitized tumor cells to T cell killing. Mechanistically, Pygo2 orchestrated a p53/Sp1/Kit/Ido1 signaling network to foster a microenvironment hostile to CTLs. Genetic or pharmacological inhibition of Pygo2 enhanced the antitumor efficacy of immunotherapies using immune checkpoint blockade (ICB), adoptive cell transfer, or agents inhibiting myeloid-derived suppressor cells. In human prostate cancer samples, Pygo2 expression was inversely correlated with the infiltration of CD8 + T cells. Analysis of the ICB clinical data showed association between high PYGO2 level and worse outcome. Together, our results highlight a potential path to improve immunotherapy using Pygo2-targeted therapy for advanced prostate cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered ‘non-immunogenic’ with trials demonstrating its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and anti-tumor immunity, characterized by modulating T cell subsets with anti-tumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the presence of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor, targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease.

Heart failure remains among the most common and morbid health conditions. The Heart Failure Strategically Focused Research Network (HF SFRN) was funded by the American Heart Association to facilitate collaborative, high‐impact research in the field of heart failure across the domains of basic, clinical, and population research. The Network was also charged with developing training opportunities for young investigators. Four centers were funded in 2016: Duke University, University of Colorado, University of Utah, and Massachusetts General Hospital‐University of Massachusetts. This report summarizes the aims of each center and major research accomplishments, as well as training outcomes from the HF SFRN.

SHP2 inhibitors (SHP2i) alone and in various combinations are being tested in multiple tumors with overactivation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages, induced tumor-intrinsic CCL5/CXCL10 secretion, and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non–small cell lung cancer (NSCLC). However, treatment also increased intratumor granulocytic myeloid-derived suppressor cells (gMDSC) via tumor-intrinsic, NFκB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C inhibitor trials. Combined SHP2 (SHP099)/CXCR1/2 (SX682) inhibition depleted a specific cluster of S100a8/9hi gMDSCs, generated Klrg1+ CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras- and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in patients with NSCLC. Significance: Our study shows that inhibiting the SHP2/RAS/ERK pathway triggers NFκB-dependent upregulation of CXCR2 ligands and recruitment of S100A8hi gMDSCs, which suppress T cells. Combining SHP2/CXCR2 inhibitors blocks gMDSC immigration, resulting in enhanced Th1 polarization, induced CD8+KLRG1+ effector T cells with high cytotoxic activity, and improved survival in multiple NSCLC models. This article is highlighted in the In This Issue feature, p. 1

Greyscale and color doppler ultrasound are the standard when investigating testicular lesions. However, they cannot provide information on the microvasculature. As such, differentiating benign from malignant tumors is challenging. Contrast-enhanced ultrasound (CEUS) uses ultrasound contrast agents that are exclusively intravascular to image the microvasculature. This review assessed CEUS outcomes in investigating intratesticular lesions and concluded that qualitative CEUS provides the most advantage by confirming vascularity, thus differentiating non-neoplastic from neoplastic lesions with added confidence, allowing for appropriate management. Whilst results for quantitative CEUS are promising in distinguishing malignant from benign neoplasms, further research on its benefits is required.

The noninflamed microenvironment in prostate cancer represents a barrier to immunotherapy. Genetic alterations underlying cancer cell-intrinsic oncogenic signaling have emerging roles in shaping the immune landscape. Recently, we identified Pygopus 2 (PYGO2) as the driver oncogene for the amplicon at 1q21.3. Here, using transgenic mouse models of metastatic prostate adenocarcinoma, we found that Pygo2 deletion decelerated tumor progression, diminished metastases, and extended survival. Pygo2 loss augmented the infiltration of cytotoxic T lymphocytes (CTLs) and sensitized tumor cells to T cell killing. Mechanistically, Pygo2 orchestrated a p53/Sp1/Kit/Ido1 signaling network to foster a microenvironment hostile to CTLs. Genetic or pharmacological inhibition of Pygo2 enhanced the anti-tumor efficacy of immunotherapies using immune checkpoint blockade, adoptive cell transfer, or myeloid-derived suppressor cell inhibitors. In human prostate cancer samples, Pygo2 expression was inversely correlated with CD8+ T cells. Our results highlight a promising path to improving immunotherapy with targeted therapy for lethal prostate cancer.

There is growing interest in the use of contrast-enhanced ultrasound (CEUS) in diagnostic and interventional radiology. CEUS applications in interventional radiology are performed with intravascular or intracavitary administration of microbubble-based US contrast agents to allow for real-time evaluation of their distribution within the vascular bed or in body cavities, respectively, providing additional information beyond gray-scale US alone. The most common interventional-radiology-related CEUS applications in children have been extrapolated from those in adults, and they include the use of CEUS to guide lesion biopsy and to confirm drain placement in pleural effusions and intra-abdominal fluid collections. Other applications are emerging in interventional radiology for use in adults and children, including CEUS to optimize sclerotherapy of vascular malformations, to guide arthrography, and for lymphatic interventions. In this review article we present a wide range of interventional-radiology-related CEUS applications, emphasizing the current and potential uses in children. We highlight the technical parameters of the CEUS examination and discuss the main imaging findings.

In pediatric and adult populations, intravenous contrast-enhanced ultrasound (CEUS) remains off-label for imaging of organs other than the liver and heart. This limited scope inhibits potential benefits of the new modality from a more widespread utilization. Yet, CEUS is potentially useful for imaging small organs such as the thyroid gland, lymph nodes, testes, ovaries and uterus, with all having locations and vasculature favorable for this type of examination. In the adult population, the utility of CEUS has been demonstrated in a growing number of studies for the evaluation of these small organs. The aim of this article is to present a review of pediatric CEUS of the thyroid gland, lymph nodes, testes, ovaries and uterus as well as to draw from the adult literature indications for possible applications in children. Supplementary Information The online version contains supplementary material available at 10.1007/s00247-021-05006-x.

Triple-negative breast carcinoma (TNBC) is one of the most aggressive types of solid-organ cancers. While immune checkpoint blockade (ICB) therapy has significantly improved outcomes in certain types of solid-organ cancers, patients with immunologically cold TNBC are afforded only a modest gain in survival by the addition of ICB to systemic chemotherapy. Thus, it is urgently needed to develop novel effective therapeutic approaches for TNBC. Utilizing the 4T1 murine model of TNBC, we developed a novel combination immunotherapeutic regimen consisting of intratumoral delivery of high-mobility group nucleosome binding protein 1 (HMGN1), TLR2/6 ligand fibroblast-stimulating lipopeptide (FSL-1), TLR7/8 agonist (R848/resiquimod), and CTLA-4 blockade. We also investigated the effect of adding SX682, a small-molecule inhibitor of CXCR1/2 known to reduce MDSC trafficking to tumor microenvironment, to our therapeutic approach. 4T1-bearing mice responded with significant tumor regression and tumor elimination to our therapeutic combination regimen. Mice with complete tumor regressions did not recur and became long-term survivors. Treatment with HMGN1, FSL-1, R848, and anti-CTLA4 antibody increased the number of infiltrating CD4+ and CD8+ effector/memory T cells in both tumors and draining lymph nodes and triggered the generation of 4T1-specific cytotoxic T lymphocytes (CTLs) in the draining lymph nodes. Thus, we developed a potentially curative immunotherapeutic regimen consisting of HMGN1, FSL-1, R848, plus a checkpoint inhibitor for TNBC, which does not rely on the administration of chemotherapy, radiation, or exogenous tumor-associated antigen(s).

To investigate the incidence and characteristics of deep vein thrombosis (DVT) in kidney transplantation recipients and analyze whether the anatomical side of DVT was associated with the side of the transplanted organ.A single-center retrospective medical record review of patients who received a kidney transplant between January 2004 and July 2019 and who subsequently developed DVT. Only patients who received unilateral kidney transplants were included in the study. Patients who underwent concomitant pancreatic transplants, bilateral kidney transplants, or repeat procedures were excluded.Of the 2449 kidney transplants performed during the study period, 1482 were included in the analysis (948 men [64%]; mean age 61 years). Of 606 duplex ultrasound tests, 115 results confirmed the presence of DVT. The incidence of symptomatic DVT was 4.7%. The most common time of DVT diagnosis was within four weeks after transplantation. Type 2 diabetes, heart failure, acute myocardial infarction, sepsis, chronic obstructive pulmonary disease/abnormal pulmonary function, and being confined to bed were associated with DVT after kidney transplant (allThe incidence of DVT after kidney transplant was lower than the incidence reported in the literature. Being confined to a bed may be a risk factor for DVT after transplant surgery. Kidney transplant recipients who had a positive duplex ultrasound had higher Caprini risk assessment scores than transplant recipients who had negative duplex ultrasounds. There was no correlation between the side of the DVT and the side of the transplant.

The aim of this study is to assess the additional benefit of contrast-enhanced ultrasound (CEUS) over conventional ultrasonography (US) in identifying intra-testicular abnormalities among observers of different experiences. In this study, 91 focal testicular lesions (46 neoplastic, 45 non-neoplastic) imaged with gray-scale US/Doppler US and CEUS were classified using a 5-point scale. Three experienced and four inexperienced observers rated each lesion using gray-scale/color Doppler US alone and then with the addition of CEUS. Improved diagnostic specificity and accuracy with the addition of CEUS was observed for both experienced (specificity: 71.1% vs. 59.3%, p = 0.005; accuracy: 83.5% vs. 76.9%, p = 0.003) and inexperienced observers (specificity: 75.6% vs. 51.7%, p = 0.005; accuracy: 80.2% vs. 72.0%, p0.001). Significant inter-observer variability between the experienced and inexperienced observers when assessing conventional US alone was eliminated with the addition of CEUS. CEUS improves diagnostic accuracy of focal intra-testicular lesions for both experienced and inexperienced observers and reduces inter-observer variability in inexperienced operators.

OBJECTIVES The purpose of this study was to quantify contrast-enhanced ultrasound enhancement of focal fatty sparing (FFS) and focal fatty infiltration (FFI) and compare it with adjacent liver parenchyma. METHODS This was a retrospective observational study yielding 42 cases in the last 4 years. Inclusion criteria were a focal liver lesion, adequate video availability, and an established diagnosis of FFS or FFI based on clinical or imaging follow-up or a second modality. Contrast-enhanced ultrasound examinations were performed with a standard low-mechanical index technique. Commercially available software calculated quantitative parameters for a focal liver lesion and a reference area of liver parenchyma, producing relative indices. RESULTS In total, 42 patients were analyzed (19 male) with a median age of 18 (interquartile range, 42) years and a median lesion diameter of 30 (interquartile range, 16) mm. The cohort included 26 with FFS and 16 with FFI. Subjectively assessed, 27% of FFS and 25% of FFI were hypoenhancing in the arterial phase, and 73% of FFS and 75% of FFI were isoenhancing. In the venous and delayed phases, all lesions were isoenhancing. The peak enhancement (P = .001), wash-in area under the curve (P

Background Acute Kidney Injury (AKI) is a common complication of COVID-19 critical illness but the pathophysiology is uncertain. Some evidence has indicated that a vascular aetiology may be implicated. We used contrast enhanced ultrasound (CEUS) and echocardiography to study renal perfusion and global blood flow and compared our findings with measurements taken in a group of septic shock patients and healthy volunteers. Methods Prospective case-control study. Renal perfusion variables were assessed with contrast enhanced ultrasound (CEUS); macrovascular blood flow was assessed using Doppler analysis of large renal vessels; echocardiography was used to assess right and left heart function and cardiac output. Results CEUS derived parameters were reduced in in COVID-19 associated AKI compared to healthy controls (perfusion index 3415 v 548 a.u., p = 0·001; renal blood volume 7794 v 3338 a.u., p = 0·04). Renal arterial flow quantified using time averaged peak velocity (TAPV) was also reduced compared to healthy controls (36·6 v 20·9 cm/s, p = 0.004) despite cardiac index being similar between groups (2.8 v 3.7 L/min/m2, p = 0.07). There were no differences in CEUS derived or cardiac parameters between COVID-19 and septic shock patients but patients with septic shock had more heterogeneous perfusion variables. Conclusion Both large and small vessel blood flow is reduced in patients with COVID-19 associated AKI compared to healthy controls, which does not appear to be a consequence of right or left heart dysfunction. A reno-vascular pathogenesis of COVID-19 AKI seems likely.

Although often asymptomatic and detected incidentally, varicocele is a relatively common problem in patients who seek medical attention for infertility problems. Ultrasound (US) is the imaging modality of choice for evaluation, but there is no consensus on the diagnostic criteria, classification, and examination technique. In view of this uncertainty, the Scrotal and Penile Imaging Working Group of the European Society of Urogenital Radiology (ESUR-SPIWG) undertook a systematic review of the available literature on this topic, to use as the basis for evidence-based guidelines and recommendations. This paper provides the results of the systematic review on which guidelines were constructed.

Ultrasonography (US) is the first-line modality for the evaluation of vascular pathology. Although well-established for many diseases, US has inherent limitations that can occasionally hinder an accurate diagnosis. The value of US was improved by the introduction of microbubbles as ultrasonographic contrast agents (UCAs) and the emergence of contrast-enhanced ultrasonography (CEUS), following the introduction of second-generation UCAs and the emergence of modern contrast-specific techniques. CEUS offers valuable information about vascular disease, both on a macrovascular and a microvascular level, with well-established applications for carotid disease, post-interventional follow-up of abdominal aortic aneurysms, and the assessment of portal vein thrombosis. The purpose of this review is to discuss the principles of CEUS and to present an overview of its vascular applications.

This study aimed to show the cost-effective benefits of creating a sustainable local program where Descemet membrane endothelial keratoplasty (DMEK) grafts were prepared locally instead of imported from American eye banks.Retrospective observational study.In 2018, 2 local technicians were trained to prestrip DMEK grafts in Edmonton up to 2 days before surgery when local donor tissue was available. When no local tissue was available, prestripped DMEK grafts were imported from U.S. eye banks. The total cost of locally prepared and imported DMEK grafts over 27 months was compared with the cost that otherwise would have been accrued if all DMEK grafts had been imported.Over 27 months, 82 DMEK grafts (55.3%) were prepared locally and 63 DMEK grafts (44.7%) were imported. The total cost of preparing 82 grafts locally was $9349.19. The total cost of importing 63 prestripped DMEK grafts was $282 431.52. The combined total cost of locally prepared and imported DMEK grafts was $291 780.71. The total cost that otherwise would have been incurred if every graft was imported was $632 108.64. This difference in costs was $340 327.93 over 27 months.Establishing a sustainable program to make high-quality DMEK grafts with local donor corneas is a cost-effective alternative to importing prestripped DMEK grafts in Edmonton.

BKV and BKVN are common in pediatric kidney transplant, but there is limited data on treatment approaches. Our objective was to study the prevalence of BKV and BKVN utilizing only plasma qPCR and report treatment outcomes with stepwise IR and IVIG.A retrospective study of all pediatric kidney transplants from 2013 to 2020. Excluded patients21 years at transplant and immediate graft failure. Surveillance was conducted using only plasma BK qPCR at 1, 3, 6, 9, 12, 18, and 24 months and annually. BKV defined as ≥250 copies/ml and resolution as250 copies/ml. Presumed BKVN as10 000 copies/ml despite IR; and BKVN if confirmed on histology.Fifty-six patients were included in the study; 20 (35.7%) had BKV. BKV was associated with longer duration of stent, 40 vs. 33.5 days (p = .004). Two patients (3.5%) had confirmed, and 2(3.5%) had presumed BKVN. The first-line treatment was IR in 100% of patients. BKVN confirmed and presumed received IVIG every month for six doses. Viral resolution was achieved in 70%, and no difference was noted in estimated glomerular filtration rate between BKV and non-BKV group (p = .438). There were no rejection episodes, and graft survival was 100% over median follow-up of 3 years.Plasma qPCR alone is adequate for screening and monitoring treatment of BKV and BKVN. A stepwise IR and IVIG resulted in BKV resolution in the majority of patients. Larger studies are required to study the role of IVIG in the treatment of BKVN.

A 64-year-old male, with a history of chronic urinary outflow obstruction secondary to benign prostatic hyperplasia, presented with haematuria and urinary retention following spontaneous removal of his long-term catheter. The patient was septic on admission and a CT examination of the abdomen and pelvis showed an acutely inflamed urinary bladder diverticulum and extensive intra-abdominal free air. The patient was treated medically for emphysematous cystitis centred on a perforated bladder diverticulum, which was thought to be caused by the underlying infectious/inflammatory process. Alternative aetiologies for free air in the abdomen such a traumatic bladder perforation and gastrointestinal perforation were considered and excluded. The patient responded well to medical management and was discharged after an 11 day in-patient stay.

Blood vessel abnormalization alters cancer cell metabolism and promotes cancer dissemination and metastasis. However, the biological features of the abnormalized blood vessels that facilitate cancer progression and whether they can be targeted therapeutically have not been fully investigated. Here, we found that an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), is expressed preferentially in abnormalized vessels of advanced colorectal cancers in humans and that its expression correlates negatively with long-term survival. Endothelial cell-specific deletion of Flrt2 in mice selectively pruned abnormalized vessels, resulting in a unique metabolic state termed "oxygen-glucose uncoupling," which suppressed tumor metastasis. Moreover, Flrt2 deletion caused an increase in the number of mature vessels, resulting in a significant increase in the antitumor effects of immune checkpoint blockers. Mechanistically, we found that FLRT2 forms noncanonical interendothelial adhesions that safeguard against oxidative stress through homophilic binding. Together, our results demonstrated the existence of tumor-specific interendothelial adhesions that enable abnormalized vessels to facilitate cancer aggressiveness. Targeting this type of adhesion complex could be a safe and effective therapeutic option to suppress cancer progression.

// Elgilda Musi 1 , Gary K. Schwartz 1 , 2 , Jae Hyuk Yoo 3 , Shannon J. Odelberg 3 , 4 , 6 , Dean Y. Li 3 , 5 , 6 , 7 , Michael Y. Bonner 8 , Ponniah Selvakumar 9 , Shikha Rao 8 , Linda C. Gilbert 8 , 10 , Justin Elsey 8 and Jack L. Arbiser 8 , 10 1 Department of Medicine, Columbia University Medical Center, New York, New York, USA 2 Herbert Irving Comprehensive Cancer Center, Columbia University College of Medicine, New York, New York, USA 3 Department of Medicine, Program in Molecular Medicine, University of Utah, Salt Lake City, Utah, USA 4 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah, USA 5 Department of Human Genetics, University of Utah, Salt Lake City, Utah, USA 6 Department of Internal Medicine, Division of Cardiovascular Medicine, University of Utah, Salt Lake City, Utah, USA 7 Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, USA 8 Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia, USA 9 Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 10 Veterans Affairs Medical Center, Decatur, Georgia, USA Correspondence to: Jack L. Arbiser, email: jarbise@emory.edu Keywords: melanoma; chemotherapy Received: April 09, 2019 Accepted: June 05, 2019 Published: July 09, 2019 ABSTRACT Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo . Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. Significance: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.

Background Renal patients with a tunnelled haemodialysis line are at risk of fibrin ‘sheath’ formation which can lead to occlusion. Dysfunctional lines are best treated by catheter exchange with a new subcutaneous tunnel; however, there is a risk of scarring, venous stenosis, potential loss of valuable access as well as the risk of infection. Method We report a retrospective review of our experience using tunnelled line intraluminal plasty (TuLIP) in 11 patients over 16 months with fibrin sheath formation on pre-existing tunnelled haemodialysis catheters. Result All patients responded well to treatment with median line patency post TuLIP reaching 112 days. Conclusion TuLIP may have a role in extending catheter lifespan and delaying more invasive intervention.

Promoting intestinal epithelial regeneration remains a major medical challenge. Female patients taking nonsteroidal anti-inflammatory drugs are less likely to have upper gastrointestinal bleeding and ulcers than males. Using a nonsteroidal anti-inflammatory drug-induced intestinal damage mouse model, we verified that female mice recover faster than males following acute intestinal insult. Using ex vivo intestinal organoid cultures, we showed that estrogen is necessary and sufficient in enhancing the female organoid formation from breached isolated crypts via the estrogen receptor β receptor. Thus, estrogen promotes female intestinal epithelial organoid regeneration to lower the incidence of intestinal bleeding and ulceration. Animal studies were approved by University of Utah IACUC under protocol number 16-05012 and 18-02010. Key words: estrogen; non-steroidal anti-inflammatory drugs; regeneration; small bowel; stem cells; upper gastrointestinal bleeding

Clinical trials of SHP2 inhibitors (SHP2i) alone and in various combinations are ongoing for multiple tumors with over-activation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non-small cell lung cancer (NSCLC). However, treatment also increased intratumor gMDSCs via tumor-intrinsic, NF-kB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C-inhibitor trials. Combined SHP2(SHP099)/CXCR1/2(SX682) inhibition depleted a specific cluster of S100a8/9high gMDSCs, generated Klrg1+ CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras-and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in NSCLC patients.Statement of SignificanceOur study shows that inhibiting the SHP2/RAS/ERK pathway triggers NF-kB-dependent up-regulation of CXCR2 ligands and recruitment of S100A8high gMDSCs, which suppress T cells in NSCLC. Combining SHP2 and CXCR2 inhibitors blocks this gMDSC immigration, resulting in enhanced Th1 polarization, induction of CD8+ KLRG1+ effector T cells with high cytotoxic activity and improved survival in multiple NSCLC models.

BACKGROUND Ultrasound (US) is the primary modality for the investigation of scrotal pathology, including both intra- and paratesticular abnormalities. OBJECTIVE To describe the abnormalities of the paratesticular space. MATERIALS/METHODS The paratesticular space contains the epididymis, spermatic cord and the tunica vaginalis cavity and is affected by a variety of inflammatory or tumoral entities. Differential diagnosis based on US criteria is frequently problematic, as the findings are non-specific. RESULTS Some general rules apply: (i) unlike testicular lesions, extra-testicular entities are usually benign in the adult, (ii) the first steps to accurate diagnosis include careful localization of the lesion and assessment of its consistency (solid or cystic) and (iii) magnetic resonance imaging can be useful for further tissue characterization of lesions suspected to contain fat, but surgical biopsy will often provide the definite diagnosis. Contrast-enhanced ultrasound (CEUS) has been applied with limited experience indicating a narrow role, primarily for the differential diagnosis of echogenic cystic entities and the delineation of a necrotic abscess from a solid neoplasm. DISCUSSION The various abnormalities are discussed and illustrated. CONCLUSION This manuscript summarizes the literature on paratesticular lesions and the value of US in diagnosis.