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PDF file - 126K, Supplemental Figure S4. Increase in MEF2c IHC staining in AS and HSA primary tumors.

PDF file - 142K, Supplemental Figure S2. Reduction of phospho-ERK1/2 IHC staining of cutaneous-derived xenograft and cardiac-derived tumorgraft treated with CI-1040.

Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal–regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease. Mol Cancer Ther; 12(9); 1701–14. ©2013 AACR.

PDF file - 146K, Supplemental Figure S3. Reduction of phospho-ERK1/2 IHC staining of cutaneous-derived xenograft and cardiac-derived tumorgraft treated with sorafenib.

Background Infantile epileptic encephalopathy is a heterogeneous condition that has been associated with variants in more than 200 genes. The variability in findings and prognosis creates challenges to making the correct diagnosis and initiating the appropriate therapy. Biallelic variants in NARS2, a mitochondrial aminoacyl-tRNA synthetase gene, were recently associated with neurodegenerative disorders that include epilepsy. Methods We describe two infant brothers who presented with focal status epilepticus that progressed to lethal epileptic encephalopathy. We compared the cost of diagnostic laboratory evaluation for each child. Detailed NARS2 protein analysis was performed using a sequence-to-structure-to-function workflow, merging multiple homologous structures, to suggest biologic impact of the NARS2 variants. Results Brain magnetic resonance imaging showed rapid progression to generalized atrophy. Extensive metabolic, infectious, chromosomal and genetic testing of the first infant failed to reach a specific diagnosis. The younger brother presented similarly. Rapid whole exome sequencing was performed revealing novel biallelic variants in NARS2. The variants c.167A>G (p.Gln56Arg) and c.631T>A (p.Phe211Ile) were confirmed in a reserved sample from the older brother. Management was then redirected toward palliative care and the child died at age nine months. Conclusions NARS2-related disorder should be considered in infants presenting with refractory seizures and rapid brain atrophy. Metabolic screening tests may be normal or yield nonspecific findings. Rapid whole exome sequencing in children with fulminant onset intractable epilepsy may minimize extensive diagnostic evaluation and aid in prognosis and medical management.

Background and purpose Upon referral from the primary care provider (PCP), dementia is diagnosed either by a neuropsychological evaluation (NPE) or at a multidisciplinary neurocognitive clinic (MNC). Following the NPE, patients continue receiving care from their PCP. In contrast, patients at the MNC are followed by a multidisciplinary care team that provides expertise across specialties in dementia care and education for the patient, family members, and care providers. The purpose of the study was to determine the utilization of acute healthcare services during the 2 years following a diagnosis of dementia in patients from the MNC and NPE. Methods A retrospective review was performed of 581 electronic medical records from January 2010 through December 2014 for 2 cohorts of patients diagnosed with dementia 1) by a neuropsychologist or 2) in a MNC. Acute-care hospital admissions, emergency room (ER) visits, and nonroutine PCP visits were identified. Categorical demographics and utilization variables were summarized by frequency. Chi-square analysis was used to analyze demographic characteristics and overall utilization between MNCs and NPE. Utilization in comparison with various demographic characteristics was analyzed using Spearman correlation coefficients and negative binomial regressions. Results Patients evaluated in the MNC were older, more severely impaired, and lived alone more often compared with NPE patients, but there was no increase in hospital admissions and ER visits. Patients who underwent NPE were 1.58 times more likely to have a nonroutine PCP office visit than patients evaluated in the MNC (p=0.0093). Conclusions Performing follow-up in multidisciplinary clinics provides patients with more education and may help to reduce the utilization of healthcare services.

Angiosarcoma (AS) is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. Using tumorgraft models, we previously showed that AS is sensitive to small-molecule inhibitors that target mitogen-activated/extracellular-signal-regulated protein kinase kinases 1 and 2 (MEK). The objective of this study was to identify drugs that combine with MEK inhibitors to more effectively inhibit AS growth. We examined the in vitro synergy between the MEK inhibitor PD0325901 and inhibitors of eleven common cancer pathways in melanoma cell lines and canine angiosarcoma cell isolates. Combination indices were calculated using the Chou-Talalay method. Optimized combination therapies were evaluated in vivo for toxicity and efficacy using canine angiosarcoma tumorgrafts. Among the drugs we tested, rapamycin stood out because it showed strong synergy with PD0325901 at nanomolar concentrations. We observed that angiosarcomas are insensitive to mTOR inhibition. However, treatment with nanomolar levels of mTOR inhibitor renders these cells as sensitive to MEK inhibition as a melanoma cell line with mutant BRAF. Similar results were observed in B-Raf wild-type melanoma cells as well as in vivo, where treatment of canine AS tumorgrafts with MEK and mTOR inhibitors was more effective than monotherapy. Our data show that a low dose of an mTOR inhibitor can dramatically enhance angiosarcoma and melanoma response to MEK inhibition, potentially widening the field of applications for MEK-targeted therapy.

Early diagnosis of dementia leads to early treatment which is beneficial to patients and the community. We reviewed initial evaluations from the Spectrum Health Medical Group Neurocognitive Clinic (SHMGNC) to evaluate dementia stage at the time of diagnosis.We retrospectively reviewed 110 randomly chosen initial evaluations from September 2008 to December 2015 at the SHMGNC. Patients underwent a neurological examination, Montreal Cognitive Assessment, and a battery of neuropsychological testing.Of all, 78.9% had moderate or severe dementia at diagnosis. The SHMGNC recommended lifestyle changes (medication assistance, financial assistance, driving restrictions, and institutional care) in 75.8% of patients with dementia. The severity of dementia was associated with the number of lifestyle changes recommended. Cohabitation with a caregiver did not lead to an early diagnosis of dementia.Patients are not undergoing evaluation at the onset of the dementia process. Diagnosis is delayed. Home-based, patient-centered care may improve early screening and detection of dementia.

Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal–regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease. Mol Cancer Ther; 12(9); 1701–14. ©2013 AACR.

We examined the feasibility of a thermal imager smart phone attachment as a potential proxy of skin perfusion by assessing shifts in skin temperature following administration of the vasodilatory anesthetic propofol. Four limb distal extremity thermal images were taken before propofol administration and at 5-min intervals thereafter during monitored anesthesia. The study enrolled 60 patients with ages ranging from 1.3 to 18 years (mean 10.7 years old) from April 2016 to January 2017. Five minutes following propofol administration, the median temperature differential (delta temperature) between the core and extremity skin significantly decreased in both upper and lower extremities, 7.9 to 3.6 &deg, C (p &lt, 0.0001) and 12.1 to 6.9 &deg, 0.0001), respectively. By 10 min, the median delta temperatures further decreased significantly in the upper (p = 0.0068) and lower extremities (p = 0.0018). There was a concordant decrease in mean blood pressure (MBP). These trends reverted back when the subject awoke. There was no significant difference between the four operators who used the camera (p = 0.0831). Blood pressure and time temperature change was the only value of significance. Mobil thermal imaging represents a non-invasive modality to assess perfusion in real time. Further studies are required to validate the clinical utility.

In epithelial cells, Sec3 associates with Exocyst complexes enriched at desmosomes and centrosomes, distinct from Sec6/8 complexes at the apical junctional complex. RNAi-mediated suppression of Sec3 alters trafficking of desmosomal cadherins and impairs desmosome morphology and function, without noticeable effect on adherens junctions., The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.

Lower limb peripheral vascular disease (PVD) results from the occlusion of arteries leading to reduced blood flow and limb ischemia. Compensatory growth of blood vessels may be sufficient to overcome limb ischemia but for some patients therapeutic intervention, even limb amputation, is required. To gain insight into the mechanisms regulating compensatory growth of blood vessels we used a mouse model of hindlimb ischemia. The ischemic lesions were confirmed and compensatory arteriogenesis was evaluated by high frequency power Doppler ultrasound. In this model, compensatory growth occurs mainly through arteriogenesis. While signaling by mitogen activated protein kinase kinases 1 and 2 (MEK 1 and 2) is required for developmental and tumor angiogenesis, it is unknown whether MEK1/2 activity drives other physiologic vascular growth such as arteriogenesis. We hypothesized that MEK 1/2 activity is necessary for hindlimb re-vascularization following femoral artery ligation. To test this we ligated the femoral artery in mice and then treated them with PD0325901, an allosteric MEK1/2 inhibitor. Following femoral artery ligation active MEK signaling was detected in spindle-shaped cells located within regions of intense angiogenesis and myocyte proliferation. MEK inhibition with PD0325901 reversibly blocked neovascularization, muscle regeneration, and caused extensive coagulative necropathy. MEK inhibition not only prevented arteriogenesis in ischemic limbs but also caused a reduction in arterial diameter. Finally, MEK inhibition prevented accumulation of CD68+ cells in ischemic tissues and skewed systemic cytokine expression towards a persistent, pro-inflammatory phenotype. Our investigation provides pharmacologic evidence demonstrating an essential role for MEK signaling in angiogenesis and arteriogenesis in response to hind limb ischemia. Further, we observed activation of MEK signaling is required for accumulation of macrophages in ischemic tissues and prevents down-regulation of systemic pro-inflammatory cytokines. Lastly, since the effects of MEK inhibition on reperfusion are reversible, we can use MEK inhibitors to create a mouse model of chronic limb ischemia that mimics aspects of PVD.

The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.

‘Skepticism is a healthy response to diagnosis of any tumor as angiosarcoma. ‘ (Lane, 1952) Angiosarcoma (AS) is an aggressive malignancy of vascular tissue or vessel forming cells (Requena & Sangueza 1998). AS is rare in humans, making up 1-2 % of soft-tissue sarcomas (Young et al. 2010) and having an estimated incidence of 0.2/100,000 persons per year. Although AS can present anywhere in the body, in humans they typically arise in the skin or superficial soft tissues. It is most frequently noted on the face and scalp of elderly men where their persistent growth causes ulceration and infection, as well as on breasts, and extremities (Brennan et al. 2001; Fayette et al. 2007; Glazebrook et al. 2008). Less frequently AS arises in liver, heart, and spleen (Young et al. 2010). The literature is replete with retrospective analyses and case studies on AS but the rarity of patients diagnosed with this disease makes it difficult to perform more than a superficial investigation on the biology and clinical behavior of AS. However, the occurrence of AS is not restricted to humans and there are several alternative animal models with the potential to inform human studies. We have written this review to collect, compare, and contrast diverse reports on the biology and treatment of AS in humans and alternative animal models. Our objective is to establish a comparative framework to focus further discussion and scientific enquiry.

Mitogen activated protein kinase kinase 1/2 (MEK1/2) inhibitors have failed to demonstrate a sustained clinical benefit for the treatment of melanoma or other MEK1/2 dependent cancers. We hypothesize combined treatment with inhibitors of MEK1/2 plus another cancer pathway would provide a more durable tumor response for MEK1/2 driven cancers. To test this we examined in vitro synergy between MEK1/2 inhibitor PD0325901 and inhibitors to eleven common cancer pathways using MEK1/2-driven melanoma cell lines and canine hemangiosarcoma cell isolates. Combination indices (CI) were calculated following the methods of Chou and Talalay (Chou and Talalay, 1984). Of the combinations tested, tyrosine kinase receptor inhibitors sorafenib and dasatinib, and the dual PI3 kinase and mTOR inhibitor rapamycin showed synergy (CI Citation Format: Nicholas John Andersen, Elissa Boguslawski, Roe Froman, Nicholas Duesbery. Improved response of mitogen activated protein kinase kinase 1/2-dependent tumors using combination drug therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3270. doi:10.1158/1538-7445.AM2013-3270

Angiosarcoma is a rare endothelial derived soft-tissue tumor with an estimated incidence of 0.2/100,000 persons/year. Current treatment regimes include surgical resection followed by chemotherapy. Even after treatment, reoccurrence is likely and usually fatal. The canine equivalent of angiosarcoma, hemangiosarcoma (HSA), is a relatively common canine endothelial neoplasm with an overall incidence of 24/100,000. In addition, canine HSA is more prevalent in specific breeds suggesting a genetic component. Similar to human angiosarcoma, HSA present as two basic forms: visceral (spleen or cardiac) and dermal. Furthermore, canine HSA occur spontaneously making canine HSA a relevant model system to study human angiosarcoma. Interestingly, MEK/MAPK signaling cascade is activated in Kaposi's Sarcoma, another human endothelial derived cancer. We hypothesize that the MEK/MAPK signaling cascade is vital for HSA and angiosarcoma growth and survival. We performed expression analysis supplemented by small molecule inhibitor studies utilizing canine primary cell isolates derived from visceral and dermal HSA. MAPK pathway expression signatures are elevated in HSA primary isolates compared to proliferating endothelial cells. Consistent with mRNA expression studies, ERK2 is constitutively phosphorylated in serum starved conditions. In addition, HSA cell viability is reduced by the MEK1/2 inhibitor CI-1040 compared to isolated canine endothelial cells. Other inhibitors targeting tyrosine kinase receptors (Sorafenib, EGFR Inhibitor, and SU11652) also decrease HSA cell viability. We conclude a network of tyrosine kinase receptors constitutively activate the MEK/MAPK signaling pathway in HSA. This work provides insights in the etiology and therapeutic potential for targeting both canine HSA and angiosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2929. doi:10.1158/1538-7445.AM2011-2929