Assessment of breast pathologies using nonlinear microscopy

Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.National Institutes of Health (U.S.) (Grant R01-CA178636-01)National Institutes of Health (U.S.) (Grant R01-CA75289-16)United States. Air Force Office of Scientific Research (Grant FA9550-10-1-0551)United States. Air Force Office of Scientific Research (Grant FA9550-12-1-0499)National Institutes of Health (U.S.) (National Research Service Award Postdoctoral Fellowship F32-CA165484

The Chemokine CXCL16 and Its Receptor, CXCR6, as Markers and Promoters of Inflammation-Associated Cancers

Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes

Local Gene Silencing of Monocyte Chemoattractant Protein-1 Prevents Vulnerable Plaque Disruption in Apolipoprotein E-Knockout Mice

Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine (CCL2), has been demonstrated to play important roles in atherosclerosis and becoming an important therapeutic target for atherosclerosis. The present study was undertaken to test the hypothesis that local RNAi of MCP-1 by site-specific delivery of adenovirus-mediated small hairpin RNA (shRNA) may enhance plaque stability and prevent plaque disruption in ApoE−/− mice. We designed an adenovirus-mediated shRNA against mouse MCP-1 (rAd5-MCP-1-shRNA). Male apolipoprotein E-knockout (ApoE−/−) mice (n = 120) were fed a high-fat diet and vulnerable plaques were induced by perivascular placement of constrictive collars around the carotid artery, intraperitoneal injection of lipopolysaccharide and stress stimulation. Mice were randomly divided into RNA interference (Ad-MCP-1i) group receiving local treatment of rAd5-MCP-1-shRNA suspension, Ad-EGFP group receiving treatment of rAd5-mediated negative shRNA and mock group receiving treatment of saline. Two weeks after treatment, plaque disruption rates were significantly lower in the Ad-MCP-1i group than in the Ad-EGFP group (13.3% vs. 60.0%, P = 0.01), and local MCP-1 expression was significantly inhibited in the Ad-MCP-1i group confirmed by immunostaining, qRT-PCR and western blot (P<0.001). Compared with the Ad-EGFP group, carotid plaques in the Ad-MCP-1i group showed increased levels of collagen and smooth muscle cells, and decreased levels of lipid and macrophages. The expression of inflammatory cytokines and activities of matrix metalloproteinases (MMPs) were lower in the Ad-MCP-1i group than in the Ad-EGFP group. In conclusion, site-specific delivery of adenoviral-mediated shRNA targeting mouse MCP-1 downregulated MCP-1 expression, turned a vulnerable plaque into a more stable plaque phenotype and prevented plaque disruption. A marked suppression of the local inflammatory cytokine expression may be the central mechanism involved

The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1ter/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders

High-resolution imaging of human atherosclerotic carotid plaques with micro18F-FDG PET scanning exploring plaque vulnerability

FDG-PET can be used to identify vulnerable plaques in atherosclerotic disease. Clinical FDG-PET camera systems are restricted in terms of resolution for the visualization of detailed inflammation patterns in smaller vascular structures. The aim of the study is to evaluate the possible added value of a high-resolution microPET system in excised carotid plaques using FDG. In this study, 17 patients with planned carotid endarterectomy were included. Excised plaques were incubated in FDG and subsequently imaged with microPET. Macrophage presence in plaques was evaluated semi-quantitatively by immunohistochemistry. Plaque calcification was assessed additionally with CT and correlated to FDG uptake. Finally, FDG uptake and macrophage infiltration were compared with patient symptomatology. Heterogeneous distributions and variable intensities of FDG uptake were found within the plaques. A positive correlation between the distribution of macrophages and the FDG uptake (r = 0.68, P <.01) was found. A negative correlation was found between areas of calcifications and FDG uptake (r = -0.84, P <.001). Ratio FDG(max) values as well as degree of CD68 accumulation were significantly higher in CVA patients compared with TIA or amaurosis fugax patients (P <.05) and CVA patients compared with asymptomatic patients (P <.05). This ex vivo study demonstrates that excised carotid plaques can be visualized in detail using FDG microPET. Enhancement of clinical PET/CT resolution for similar imaging results in patients is needed

Identification of four new susceptibility loci for testicular germ cell tumour

Genome wide association studies (GWAS) have identified multiple risk loci for testicular germ cell tumour (TGCT), revealing a polygenic model of disease susceptibility strongly influenced by common variation. To identify further SNPs associated with TGCT we conducted a multistage GWAS with combined dataset of >25,000 individuals (6,059 cases and 19,094 controls). We identified new risk loci for TGCT at 3q23 (rs11705932, TFDP2, P = 1.5 x 10-9), 11q14.1 (rs7107174, GAB2, P = 9.7 x 10-11), 16p13.13 (rs4561483, GSPT1, P = 1.6 x 10-8) and 16q24.2 (rs55637647, ZFPM1, P = 3.4 x 10-9). We additionally present detailed functional analysis of these loci, identifying a strong relationship between rs4561483 risk genotype and increased GSPT1 expression in TGCT patient samples. These findings provide additional support for a polygenic model of TGCT risk and further insight into the biologic basis of disease development

CXCR3 controls T cell accumulation in fat accumulation

OBJECTIVE - Obesity associates with increased numbers of inflammatory cells in adipose tissue (AT), including T cells, but the mechanism of T-cell recruitment remains unknown. This study tested the hypothesis that the chemokine (C-X-C motif) receptor 3 (CXCR3) participates in T-cell accumulation in AT of obese mice and thus in the regulation of local inflammation and systemic metabolism. APPROACH AND RESULTS - Obese wild-type mice exhibited higher mRNA expression of CXCR3 in periepididymal AT-derived stromal vascular cells compared with lean mice. We evaluated the function of CXCR3 in AT inflammation in vivo using CXCR3-deficient and wild-type control mice that consumed a high-fat diet. Periepididymal AT from obese CXCR3-deficient mice contained fewer T cells than obese controls after 8 and 16 weeks on high-fat diet, as assessed by flow cytometry. Obese CXCR3-deficient mice had greater glucose tolerance than obese controls after 8 weeks, but not after 16 weeks. CXCR3-deficient mice fed high-fat diet had reduced mRNA expression of proinflammatory mediators, such as monocyte chemoattractant protein-1 and regulated on activation, normal T cell expressed and secreted, and anti-inflammatory genes, such as Foxp3, IL-10, and arginase-1 in periepididymal AT, compared with obese controls. CONCLUSIONS - These results demonstrate that CXCR3 contributes to T-cell accumulation in periepididymal AT of obese mice. Our results also suggest that CXCR3 regulates the accumulation of distinct subsets of T cells and that the ratio between these functional subsets across time likely modulates local inflammation and systemic metabolism. © 2014 American Heart Association, Inc.link_to_OA_fulltex