Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.

Context Cushing’s syndrome (CS) is associated with impaired health-related quality of life (HRQOL) even after surgical cure. Objective To characterize patient and provider perspectives on recovery from CS, drivers of decreased HRQOL during recovery, and ways to improve HRQOL. Design Cross-sectional observational survey Participants Patients (n=341) had undergone surgery for CS and were members of the Cushing’s Support and Research Foundation. Physicians (n=54) were Pituitary Society physician members and academicians who treated patients with CS. Results Compared to patients, physicians underestimated the time to complete recovery after surgery (12 months vs 18 months, p=0.0104). Time to recovery did not differ by CS etiology, but patients with adrenal etiologies of CS reported a longer duration of cortisol replacement medication (CRM) compared to patients with Cushing’s disease (12 months vs 6 months, p=0.0025). Physicians overestimated the benefits of work (26.9% vs 65.3%, p <0.0001), exercise (40.9% vs 77.6%, p=0.0001), and activities (44.8% vs 75.5%, p=0.0016) as useful coping mechanisms in the post-surgical period. Most patients considered family/friends (83.4%) and rest (74.7%) to be helpful. All physicians endorsed educating patients on recovery, but 32.4% (95% CI 27.3%-38.0%) of patients denied receiving sufficient information. Some patients did not feel prepared for the post-surgical experience (32.9%, 95% CI 27.6%-38.6%) and considered physicians not familiar enough with CS (16.1%, 95% CI 12.2%-20.8%). Conclusion Poor communication between physicians and CS patients may contribute to dissatisfaction with the post-surgical experience. Increased information on recovery, including helpful coping mechanisms, and improved provider-physician communication may improve HRQOL during recovery.

Health systems are struggling to manage a fluctuating volume of critically ill patients with COVID-19 while continuing to provide basic surgical services and expand capacity to address operative cases delayed by the pandemic. As we move forward through the next phases of the pandemic, we will need a decision-making system that allows us to remain nimble as clinicians to meet our patient’s needs while also working with a new framework of healthcare operations. Here, we present our quality improvement process for the adaptation and application of the Medically Necessary Time-Sensitive (MeNTS) toolto gynecologic surgical services beyond the initial COVID response and into recovery of surgical services; with analysis of the reliability of the modified-MeNTS tool in our multi-site safety net hospital network. This multicenter study evaluated the gynecology surgical case volume at three tertiary acute care safety net institutions within the LA County Department of Health Services: Harbor-UCLA (HUMC), Olive View Medical Center (OVMC), and Los Angeles County + University of Southern California (LAC+USC). We describe our modified-Delphi approach to adapt the MeNTS tool in a structured fashion and its application to gynecologic surgical services. Blinded reviewers engaged in a three-round iterative adaptation and final scoring utilizing the modified tool. The cohort consisted of 392 female consecutive gynecology patients across three Los Angeles County Hospitals awaiting scheduled procedures in the surgical queue.The majority of patients were Latina (74.7%) and premenopausal (67.1%). Over half (52.4%) of the patients had cardiovascular disease, while 13.0% had lung disease, and 13.8% had diabetes. The most common indications for surgery were abnormal uterine bleeding (33.2%), pelvic organ prolapse (19.6%) and presence of an adnexal mass (14.3%). Minimally invasive approaches via laparoscopy, robotic-assisted laparoscopy, or vaginal surgery was the predominant planned surgical route (54.8%). Modified-MeNTS scores assumed a normal distribution across all patients within our cohort (Median 33, Range 18–52). Overall, ICC across all three institutions demonstrated “good” interrater reliability (0.72). ICC within institutions at HUMC and OVMC were categorized as “good” interrater reliability, while LAC-USC interrater reliability was categorized as “excellent” (HUMC 0.73, OVMC 0.65, LAC+USC 0.77). The modified-MeNTS tool performed well across a range of patients and procedures with a normal distribution of scores and high reliability between raters. We propose that the modified-MeNTS framework be considered as it employs quantitative methods for decision-making rather than subjective assessments.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

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Ewing sarcoma (ES) is a pediatric tumor induced by EWS-ETS fusion proteins, most often EWS-FLI1. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear. Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of nonperfused areas within tumor tissue was associated with poor prognosis. In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes via changes in transcriptional activity of the EWS-FLI1 gene. However, despite this line of evidence, no direct proof for this hypoxia-induced ES progression and spread has been provided. Moreover, the mechanisms by which hypoxia could exert such effects are unknown. To fill this gap, we created an in vivo model of hypoxia in ES and tested its effect on tumor metastasis. SK-ES1 ES cells were injected into gastrocnemius muscles of SCID/beige mice. Once the tumors reached a volume of 250mm3, they were either excised (control) or subjected to femoral artery ligation (FAL) for 72h prior to excision, inducing ischemia of the lower hindlimb, thus creating tumor hypoxia. Then, the mice were monitored for metastases. The extent of the metastatic disease was assessed and compared between experimental groups based on periodic MRI, necropsy and histopathology findings. FAL resulted in profound tumor hypoxia, as evidenced by inhibition of primary tumor growth, severe tissue necrosis and positive staining for a hypoxyprobe, pimonidazole. However, despite the impaired growth of primary tumors, xenografts subjected to FAL were more metastatic. The involvement of hypoxic cells in metastases was evidenced by the accumulation of pimonidazole-positive cells (hypoxic at the time of FAL) in areas of tissue invasion and intravasation. Consequently, mice bearing FAL-treated tumors exhibited a decreased latency of metastases formation and an increase in their number from an average of 0.9 to 2.3 metastases per mouse in control and FAL groups, respectively. We also observed a change in the pattern of metastases, as FAL-treated tumors metastasized more often to distant organs (average of 0.3 organ metastases per mouse in control and 1.3 in FAL group). The hypoxia-induced metastases were most often observed in adrenal gland and spine (50% and 42% of mice in FAL group, respectively), while no such metastases were observed in the control group. Moreover, 100% of FAL-treated mice had signs of bone marrow invasion, while no tumor cells were detectable in bone marrow of control mice. This data provides the first-ever direct evidence for tumor hypoxia as a driver of ES metastases. Moreover, our model of tumor hypoxia in vivo provides an excellent opportunity to identify hypoxia-induced pathways involved in ES metastatic progression that subsequently may become novel therapeutic targets for this disease. Citation Format: Jason U. Tilan, Sung-Hyeok Hong, Susana Galli, Rachel Acree, Katherine Connors, Meredith Horton, Akanksha Mahajan, Larissa Wietlisbach, Yi-Chien Lee, Olga Rodriguez, Christopher Albanese, Joanna Kitlinska. Tumor hypoxia promotes Ewing sarcoma metastases in a mouse xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2478.

Ewing sarcoma (ES) is a tumor driven by EWS-ETS fusion proteins. Yet, the same fusions are present in localized and metastatic tumors that carry strikingly different prognoses. Despite low levels of genomic instability in primary ES tumors, the presence of complex karyotypes is one of a few adverse prognostic factors, implicating an acquired chromosomal instability (CIN) in ES progression. As transcriptional targets of EWS-ETS, neuropeptide Y (NPY) and its Y5 receptor (Y5R) are highly expressed in ES and further activated by hypoxia. We have found that overexpression of Y5R leads to defects in cytokinesis, followed by formation of polyploid cells, chromosome loss and CIN. Thus, the goal of our study was to determine whether CIN that is driven by hypoxia-induced activation of NPY/Y5R axis promotes ES metastases. ES cells were injected into gastrocnemius muscles of SCID/beige mice. Hypoxia in the resulting primary tumors was created by 72h ligation of the femoral artery. Then, the tumors were excised and mice were monitored for metastases. Tissues and cells derived from primary tumors and metastases were subjected to cytogenetic analyses. ES metastasis was associated with progressive genomic changes in tumor cells. Cells derived from primary tumors exhibited increases in nuclear sizes and ploidy, as compared to the original cells. Tumor hypoxia exacerbated this effect. This initial increase in ploidy was followed by a decrease in nuclear size, increase in mitotic errors and reduced chromosome numbers in cells from metastatic tissues, suggesting that ES progression associates with increased CIN and is triggered by cell polyploidization. This notion was confirmed by increased DNA copy number alterations in tissues from ES metastases observed in xenografts derived from 2 different cell lines and a clinical case of matched primary tumor and metastasis tissue (array-CGH). In SK-ES1 xenografts, these alterations involved gains in the locus of Y5R. Consequently, FISH identified an SK-ES1 clone with 3 copies of the Y5R gene. The percent of cells with Y5R gene amplification increased with the degree of SK-ES1 progression, with 16-24% cells in the original SK-ES1 cell line, 40-60% in primary tumors and 86-100% in metastases. This was associated with an increase in Y5R expression in metastatic tissues. Thus, the metastasis in SK-ES1 xenografts associated with a selection of the clone with amplified Y5R. SK-ES1 cells subjected to hypoxia in vitro presented with similar increases in nuclear sizes and enrichment in the clone with amplified Y5R (48%), as was observed in primary tumors. Y5R activation in normoxic SK-ES1 cells mimicked this effect. Our findings support the role for acquired CIN in ES progression and metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. Thus, Y5R antagonist may serve as an adjuvant treatment to prevent ES CIN and progression. Citation Format: Akanksha Mahajan, Sung-Hyeok Hong, Jason U. Tilan, Susana Galli, Congyi Lu, Jasmine Rodgers, Anju Duttargi, Rachel Acree, Luciane R. Cavalli, Joanna B. Kitlinska. Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2443.

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