Citations

Abstract The management of systemic amyloidosis relies on the treatment of the underlying etiology and differs radically for different amyloid types. Therefore, given that at least 25 different proteins have been associated with amyloidosis, accurate identification of proteins deposited as amyloid fibrils is an important clinical problem. In this study, we describe a novel method that can characterize amyloid subtypes using laser microdissection (LMD) and mass spectrometry (MS) on routinely processed paraffin-embedded tissues. The study used 60 cases consisting of 16 transthyretin, 9 serum amyloid-associated protein, 20 immunoglobulin light chain lambda, 5 immunoglobulin light chain kappa, and 10 amyloid negative control samples. The biopsy specimens studied included heart, kidney, gastrointestinal tract, lung and decalcified bone marrow specimens. The amyloid type in all cases was previously characterized based on clinical findings, immunohistochemistry and, where indicated, by molecular testing for transthyretin mutations. Amyloid plaques were captured from an 10 micron paraffin section exhibiting positive Congo Red staining using LMD. Proteins were extracted, digested with trypsin and identified following MS/MS using the Mascot search algorithm analysis. MS correctly identified each of the 4 types of amyloidosis analyzed. Serum Amyloid P component and Apolipoprotein E were also identified as constituents of the amyloid deposition. The analysis was successful on all tissue types including decalcified bone marrow specimens and small biopsy specimens such as endomycardial biopsies and renal biopsies. The use of LMD from paraffin embedded biopsies and subsequent analysis by MS allows identification of the type of amyloid protein deposited with high specificity and sensitivity. This method promises to be a clinical test for accurate identification of amyloid proteins in routinely processed biopsy specimens and overcomes many of the specificity and sensitivity issues associated with current methods such as immunohistochemistry.

Abstract Angiogenesis plays an important role in tumor growth. Endothelial cells express the chemokine receptor CXCR4, and interact with its ligand SDF-1/CXCL12. Previous studies have demonstrated that the ERK MAP kinase and the PI3Kinase pathways are activated in response to SDF-1 stimulation. In this study, we investigate the effect of the role of inhibitors of CXCR4, ERK MAP kinase and PI3Kinase on angiogenesis. The AngioKit (TCS Cellworks, U.K) is a 24 well plate in which human endothelial cells are co-cultured with other human myoblasts and fibroblasts in a specially designed medium. Control wells in the kit include media alone, VEGF (+control) and suramin (-control). Test samples were added on the day the kits arrive, then changed on days 4, 7, and 9, and stained on day 11 with CD31 (PECAM). The wells are then photographed and subjected to image analysis. The software measures angiogenesis as total tubule length per well in microns. Test samples can then be compared to the control wells to determine the drugs affect on angiogenesis in vitro. The following drugs were tested in this angiogenesis model system, a human CXCR4 neutralizing antibody (MAB 171, R&D systems, MN), SDF-1, the MAP kinase inhibitor PD098059, and the PI3Kinase inhibitor LY294002. Treatment with the CXCR4 inhibitory antibody, PD098059, and LY294002 caused marked decrease in angiogenesis (below the level of the negative control suramin). Inhibition of angiogenesis below the level of suramin was first detected at 1mg/ml CXCR4 antibody, and10mg/ml CXCR4 antibody resulted in complete inhibition of angiogenesis. The effect of PD098059 on angiogenesis was dependent on its concentration; 20mM PD098059 inhibited angiogenesis while lower concentrations did not. These results are consistent with the drug’s known concentration-dependent inhibition of MEK-1 and indicate that the MEK-1 inhibitor leads to angiostasis secondary to its specific inhibitory effect on MEK-1. The lowest level tested of 1μM LY294002 led to inhibition of angiogenesis, and 50μM of LY294002 led to complete abrogation of angiogenesis. SDF-1 has been reported to be angiogenic. In this model system, the effect of SDF-1 alone on angiogenesis was subtle. However, the endothelial cells used in this model system may be secreting endogenous SDF-1 leading to the saturation of the CXCR4 receptor and minimal effects of exogenous SDF-1 stimulation. This was demonstrated by the significant effect of the CXCR4 inhibitor on angiogenesis without the addition of exogenous SDF-1. These results indicate that CXCR4 inhibition and its downstream pathways PI3K and ERK MAP kinase lead to significant inhibition of angiogenesis, and suggest that selective inhibitors of CXCR4 may be useful agents to inhibit angiogenesis.

Aim The aim of this is to identify the gene expression profiles indicative of reversible versus irreversible injury in experimental (murine) renal artery stenosis (RAS). Method Two‐kidney‐one‐cuff hypertension was established by placement of a polytetrafluoroethylene cuff on the right renal artery (N=18). Sham surgery was performed by manipulating the right renal artery without cuff placement (N=6). To determine potential reversibility, the cuff was removed at 7 days (7D cuff‐off, N=6) and 14 days (14D cuff‐off, N=6) after initial RAS surgery. In a control RAS group, the cuff was left in place for 28 days (28D RAS, N=6). Kidneys were harvested at 28 days for assessment of atrophy and fibrosis. Renal blood perfusion and oxygenation were measured using 16.4T MRI. RNASeq analysis was performed on all the groups at 28 days following initial surgery. Results Following cuff placement, intra‐renal perfusion was reduced by 65% (p<0.0001 vs. baseline). In the 7D cuff‐off group intra‐renal perfusion was restored to baseline values after cuff removal, whereas in the 14D cuff‐off group perfusion was not significantly improved following cuff removal. In the 28D RAS group the reduction in perfusion was maintained throughout the study period. The 7D cuff‐off group had a significantly lower % atrophy (7 ± 2.2%, p=0.0042) and % area of interstitial fibrosis (0.57 ± 0.08 %, p<0.0001) compared to 28D RAS group (atrophy 66 ± 13%, area of fibrosis 7.79 ± 0.89 %). The 14D cuff‐off group had severe atrophy (56 ± 16.6%) and interstitial fibrosis (5.05 ± 1.22%) similar to that seen in 28D RAS group. RNASeq analysis data were normalized to appropriate sham controls in all the groups and was performed at 28 days following initial surgery. Induction of greater than Log2 ratio >2 and p values < 0.00001 were considered significant (following adjustment for multiple comparisons). Fibrosis associated genes were differentially regulated in the 7D cuff‐off compared to the 14D cuff‐off and 28D RAS group (table ). Conclusion We conclude that cuff removal at 7 days is associated with relative preservation of renal structure, indicating that renal injury at this point is reversible. We have identified a number of fibrosis associated genes that are differentially regulated in the 7D cuff‐off group versus 14D cuff‐off group. Further analysis will determine the functional relevance of these differentially regulated genes. Support or Funding Information This project was funded by National Institutes of Health, NIAID R01 AI100911. Values of Log 2 Ratio Genes 7D Cuff‐off vs Sham 14D Cuff‐off vs Sham 28D RAS vs Sham COL1A1 0.96 2.62 2.23 COL1A2 0.79 2.07 1.91 COL3A1 1.26 2.60 2.35 MMP12 1.24 2.85 2.95 MMP14 1.34 2.70 2.55 MMP19 2.89 3.86 4.05 MMP2 1.55 3.49 3.22 MMP7 6.96 9.22 9.59 MTOR −0.46 −1.67 −2.06 SERPINE1 1.25 3.37 2.35 TGFB1i1 0.44 1.46 1.53 TGFB2 0.82 1.83 1.97 TGFB3 0.59 1.91 1.99 TGFBi 0.98 1.88 2.02 KLF15 −0.90 −1.76 −2.60 UBD 4.49 6.23 6.18 TIMP1 2.94 5.30 5.16 TIMP2 0.50 1.46 1.52 TIMP3 −0.35 −1.26 −1.36 HSPB1 0.16 2.00 2.13 HSPB8 1.51 2.26 2.30 CCL2 3.44 4.89 4.31 CCL12 2.88 3.71 4.12 SERPINE1 1.25 3.37 2.35 SERPINE2 1.17 2.39 2.35 SERPINF1 2.02 2.76 3.11 SERPINF2 −0.51 −2.03 −2.71 SERPING1 1.15 1.81 2.09 CLU 1.45 2.54 3.07 STC1 0.45 1.99 2.12 SPP1 1.75 3.06 3.37

Background: Atrial fibrillation (AF) adversely impacts health-related quality of life (hrQoL). While some patients demonstrate improvements in hrQoL, the factors associated with large improvements in hrQoL are not well described. Methods: We assessed factors associated with a 1-year increase in the Atrial Fibrillation Effect on Quality-of-Life score of 1 SD (≥18 points; 3× clinically important difference), among outpatients in the Outcomes Registry for Better Informed Treatment of Atrial Fibrillation I registry. Results: Overall, 28% (181/636) of patients had such a hrQoL improvement. Compared with patients not showing large hrQoL improvement, they were of similar age (median 73 versus 74, P =0.3), equally likely to be female (44% versus 48%, P =0.3), but more likely to have newly diagnosed AF at baseline (18% versus 8%; P =0.0004), prior antiarrhythmic drug use (52% versus 40%, P =0.005), baseline antiarrhythmic drug use (34.8% versus 26.8%, P =0.045), and more likely to undergo AF-related procedures during follow-up (AF ablation: 6.6% versus 2.0%, P =0.003; cardioversion: 12.2% versus 5.9%, P =0.008). In multivariable analysis, a history of alcohol abuse (adjusted OR, 2.41; P =0.01) and increased baseline diastolic blood pressure (adjusted OR, 1.23 per 10-point increase and >65 mm Hg; P =0.04) were associated with large improvements in hrQoL at 1 year, whereas patients with prior stroke/transient ischemic attack, chronic obstructive pulmonary disease, and peripheral arterial disease were less likely to improve ( P <0.05 for each). Conclusions: In this national registry of patients with AF, potentially treatable AF risk factors are associated with large hrQoL improvement, whereas less reversible conditions appeared negatively associated with hrQoL improvement. Understanding which patients are most likely to have large hrQoL improvement may facilitate targeting interventions for high-value care that optimizes patient-reported outcomes in AF. Registration: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01165710.

Objectives: To understand from the perspective of patients who did, and did not attend ICU recovery programs, what were the most important components of successful programs and how should they be organized. Design: International, qualitative study. Setting: Fourteen hospitals in the United States, United Kingdom, and Australia. Patients: We conducted 66 semi-structured interviews with a diverse group of patients, 52 of whom had used an ICU recovery program and 14 whom had not. Interventions: None. Measurements and Main Results: Using content analysis, prevalent themes were documented to understand what improved their outcomes. Contrasting quotes from patients who had not received certain aspects of care were used to identify perceived differential effectiveness. Successful ICU recovery programs had five key components: 1) Continuity of care; 2) Improving symptom status; 3) Normalization and expectation management; 4) Internal and external validation of progress; and 5) Reducing feelings of guilt and helplessness. The delivery of care which achieved these goals was facilitated by early involvement (even before hospital discharge), direct involvement of ICU staff, and a focus on integration across traditional disease, symptom, and social welfare needs. Conclusions: In this multicenter study, conducted across three continents, patients identified specific and reproducible modes of benefit derived from ICU recovery programs, which could be the target of future intervention refinement.

Several new systemic therapy options have become available for patients with metastatic breast cancer, which have led to improvements in survival. In addition to patient and clinical factors, the treatment selection primarily depends on the tumor biology (hormone-receptor status and HER2-status). The NCCN Guidelines specific to the workup and treatment of patients with recurrent/stage IV breast cancer are discussed in this article.

Background: Nonrheumatic valvular diseases are common; however, no studies have estimated their global or national burden. As part of the Global Burden of Disease Study 2017, mortality, prevalence, and disability-adjusted life-years (DALYs) for calcific aortic valve disease (CAVD), degenerative mitral valve disease, and other nonrheumatic valvular diseases were estimated for 195 countries and territories from 1990 to 2017. Methods: Vital registration data, epidemiologic survey data, and administrative hospital data were used to estimate disease burden using the Global Burden of Disease Study modeling framework, which ensures comparability across locations. Geospatial statistical methods were used to estimate disease for all countries, because data on nonrheumatic valvular diseases are extremely limited for some regions of the world, such as Sub-Saharan Africa and South Asia. Results accounted for estimated level of disease severity as well as the estimated availability of valve repair or replacement procedures. DALYs and other measures of health-related burden were generated for both sexes and each 5-year age group, location, and year from 1990 to 2017. Results: Globally, CAVD and degenerative mitral valve disease caused 102 700 (95% uncertainty interval [UI], 82 700–107 900) and 35 700 (95% UI, 30 500–42 500) deaths, and 12.6 million (95% UI, 11.4 million–13.8 million) and 18.1 million (95% UI, 17.6 million–18.6 million) prevalent cases existed in 2017, respectively. A total of 2.5 million (95% UI, 2.3 million–2.8 million) DALYs were estimated as caused by nonrheumatic valvular diseases globally, representing 0.10% (95% UI, 0.09%–0.11%) of total lost health from all diseases in 2017. The number of DALYs increased for CAVD and degenerative mitral valve disease between 1990 and 2017 by 101% (95% UI, 79%–117%) and 35% (95% UI, 23%–47%), respectively. There is significant geographic variation in the prevalence, mortality rate, and overall burden of these diseases, with highest age-standardized DALY rates of CAVD estimated for high-income countries. Conclusions: These global and national estimates demonstrate that CAVD and degenerative mitral valve disease are important causes of disease burden among older adults. Efforts to clarify modifiable risk factors and improve access to valve interventions are necessary if progress is to be made toward reducing, and eventually eliminating, the burden of these highly treatable diseases.

AbstractA novel group of S100‐ and CD34‐positive spindle cell tumors with distinctive stromal and perivascular hyalinization harboring recurrent gene fusions involving kinases includingRAF1,BRAF,NTRK1/2/3, andREThave been recently reported. To our knowledge, no such cases harboringALKrearrangements have been identified. We report a previously healthy 41‐year‐old male with a 12‐cm intramuscular shoulder mass. The tumor was composed of bland‐appearing spindled to epithelioid cells, arranged in a patternless pattern in a background of loose myxoid stroma containing striking amianthoid‐like stromal collagen and perivascular rings. In accordance with the previously reported tumors, the tumor cells showed diffuse immunopositivity with S100 and CD34, while lacking SOX10 expression. Targeted RNA‐based next‐generation sequencing identified a novel serine/threonine‐protein phosphatase PP1‐beta‐catalytic subunit (PPP1CB)‐ALKfusion gene. Although ALK break‐apart was not detected by FISH, likely due to a paracentric inversion of chromosome 2, the presence of the fusion was confirmed by Sanger sequencing showing a 10‐bp linker between exon 6 ofPPP1CBand intron 19 ofALKwhile maintaining reading frame. Subsequent ALK‐1 immunostain exhibited diffuse cytoplasmic staining in the tumor cells. Our case expands the molecular genetic spectrum of the distinctive group of spindle cell tumors with CD34/S100+ immunophenotype, supporting the important role of various kinases as drivers of oncogenesis. Awareness of this entity including its unique morphologic and immunophenotypic features as well as its interchangeable kinase gene fusions is crucial for correct classification and potential targeted therapy, particularly in aggressive subsets.