Intermediate Expression Research Articles

Immunohistochemical expression of epidermal growth factor receptor (EGFR) in South Asian head and neck squamous cell carcinoma: association with various risk factors and clinico-pathologic and prognostic parameters

BackgroundIn this study, we intend to determine the immunohistochemical expression of EGFR in cases of head and neck squamous cell carcinoma and its association with prognostic clinico-pathologic features.MethodsA total of 115 cases of head and neck squamous cell carcinoma treated at Liaquat National Hospital, Karachi, Pakistan, were included in the study. Clinico-pathologic features, risk factors, and recurrence status of cases were evaluated, and EGFR immunohistochemistry was performed.ResultsIn our study, 52 cases (45.2%) of head and neck SCC were positive and 10 cases (8.7%) were focal positive for EGFR expression, while 53 cases (46.1%) were negative for EGFR expression. High EGFR expression (> 70%) was noted in 6.1% (7 cases), while 12.2% (14 cases) and 26.1% (30 cases) revealed 51–70% and 11–50% EGFR expression respectively. On the basis of intensity, strong EGFR expression was noted in 13.9% (16 cases) while 16.5% (19 cases) and 23.5% (27 cases) revealed intermediate and weak EGFR expression respectively. Significant association of EGFR expression was noted with tumor stage and disease-free survival.ConclusionWe found a significant association of EGFR expression with tumor stage and disease-free survivals, which are the most important prognostic factors in head and neck squamous cell carcinoma; therefore, EGFR expression can help as a prognostic biomarker in head and neck squamous cell carcinoma. On the other hand, we suggest that molecular studies should be performed in squamous cell carcinoma of head and neck in our setup to identify patients that can avail response from anti-EGFR therapy.

Characterization of kidney CD45intCD11bintF4/80+MHCII+CX3CR1+Ly6C- "intermediate mononuclear phagocytic cells".

Kidney immune cells play important roles in pathogenesis of many diseases, including ischemia-reperfusion injury (IRI) and transplant rejection. While studying murine kidney T cells, we serendipitously identified a kidney mononuclear phagocytic cell (MPC) subset characterized by intermediate surface expression of CD45 and CD11b. These CD45intCD11bint MPCs were further identified as F4/80+MHCII+CX3CR1+Ly6C- cells, comprising ~17% of total CD45+ cells in normal mouse kidney (P < 0.01) and virtually absent from all other organs examined except the heart. Systemic clodronate treatment had more significant depletive effect on the CD45intCD11bint population (77.3%±5.9%, P = 0.03) than on CD45highCD11b+ population (14.8%±16.6%, P = 0.49). In addition, CD45intCD11bint MPCs had higher phagocytic function in the normal kidney (35.6%±3.3% vs. 24.1%±2.2%, P = 0.04), but lower phagocytic capacity in post-ischemic kidney (54.9%±1.0% vs. 67.8%±1.9%, P < 0.01) compared to the CD45highCD11b+ population. Moreover, the CD45intCD11bint population had higher intracellular production of the pro-inflammatory tumor necrosis factor (TNF)-α (58.4%±5.2% vs. 27.3%±0.9%, P < 0.001) after lipopolysaccharide (LPS) stimulation and lower production of the anti-inflammatory interleukin (IL)-10 (7.2%±1.3% vs. 14.9%±2.2%, P = 0.02) following kidney IRI, suggesting a functional role under inflammatory conditions. The CD45intCD11bint cells increased early after IRI, and then abruptly decreased 48h later, whereas CD45highCD11b+ cells steadily increased after IRI before declining at 72h (P = 0.03). We also identified the CD45intCD11bint MPC subtype in human kidney. We conclude that CD45intCD11bint F4/80+MHCII+CX3CR1+Ly6C-population represent a unique subset of MPCs found in both mouse and human kidneys. Future studies will further characterize their role in kidney health and disease.

PO-366 Long non-coding RNAs TINCR and DANCR in urothelial carcinoma subtypes

Introduction Long non-coding RNAs (lncRNAs) are a diverse set of transcripts spanning more than 200 nucleotides. Many show a tissue-specific expression pattern and are involved in fine tuning of cell fate determination by regulating gene expression. Two lncRNAs, TINCR and DANCR, are known as important factors in keratinocyte differentiation. Upregulation of TINCR is required for terminal epidermal differentiation whereas DANCR maintains the undifferentiated state of keratinocytes. We wondered whether these lncRNAs could be involved in aberrant differentiation of urothelial carcinoma (UC) and contribute to the development a specific molecular UC subtypes with squamous features, termed Basal-Squamous-like-subtype (BASQ). We analysed lncRNA expression in a large set of UC tissues and a smaller set of bladder squamous cell carcinomas (B-SCC) and compared the results with TCGA RNA-Seq data. Material and methods LncRNA expression was measured by qRT-PCR in 161 UC, 8 normal and 12 B-SCC samples. RNA-Seq data from the TCGA cohort was accessed via the TANRIC database. Statistical comparison for differential expression between each group was done by Wilcoxon rank sum test in R. Correlation of histopathological patient data with lncRNA expression was performed using SPSS. The Broad institute Morpheus tool was used for hierachical cluster analysis of the TCGA RNA-Seq data to identify associations of lncRNA expression with UC molecular subtypes. Results and discussions Consistent with the TCGA RNA-Seq data, qRT-PCR analysis of our large tissue set revealed both TINCR and DANCR to be upregulated in UC and in B-SCC compared to benign tissues. According to the qRT-PCR data, DANCR expression was significantly elevated in non-invasive over invasive tumours. Kaplan-Meier analysis did not reveal associations of patient outcome with upregulated lncRNA expression, except that high TINCR expression was associated with worse metastasis-free survival. Hierarchical cluster analysis indicated that the BASQ subtype, which is defined by low expression of luminal marker genes (FOXA1, GATA3) and high expression of basal and squamous marker genes (KRT5, KRT6, KRT14), is also characterised by intermediate TINCR expression. Conclusion Both TINCR and DANCR expression are frequently upregulated in UC, but are not strongly associated with clinical parameters. Instead, our data support the emerging consensus that specific lncRNA expression patterns are associated with and may contribute to the characteristics of UC molecular subtypes.

PO-437 Ultrasound-triggered delivery of halogenated pyruvate analogues to breast cancer cells

Introduction Targeting the altered cancer metabolism has opened new therapeutic opportunities. Upregulation of the expression of the monocarboxylate transporter 1 (MCT1) has been shown in various cancers, including breast tumours. MCT1 is responsible for the transport of lactate and pyruvate. Lactate/pyruvate analogues, such as 3-bromopyruvate, have been shown to be toxic to cells overexpressing MCT1. However, in order to successfully treat the tumour, the therapeutic must be shielded from the immune system, target the tumour and penetrate it. To overcome these challenges we have encapsulated lactate/pyruvate analogues inside ultrasound labile liposomes. These will protect the therapeutic and extend its circulation time, enabling delivery and triggered release at the tumour site using externally applied ultrasound, whereas MCT1 expression offers selective uptake of the compounds. Material and methods Western blotting was used to identify cell lines with high and low expression of MCT1. Cell viability after treatment was tested with an MTT assay. Chromatography techniques were used to measure encapsulation efficiency into the liposomes. A novel iodinated lactate analogue, 3-iodolactate (3IL), was synthesised via Finkelstein reaction and characterised by NMR spectroscopy and mass spectrometry. Ultrasound-labile liposomes were synthesised by dispersion of a lyophilised lipid film (DOPE, cholesterol and DSPC-PEG(2000) in 65:25:10 molar ratio) in an aqueous solution of the compound, followed by extrusion and size exclusion chromatography. Results and discussions Breast cancer cell lines with low, intermediate and high expression of MCT1 have been identified (MDA-MB-231, MDA-MB-468 and BT20, respectively). 3 BP toxicity correlated with MCT1 expression. BT20 had an LD50 of 87.10±0.03 µM, while MDA-MB-231, were 90% viable even up to concentrations of 300 µM 3 BP. Toxicity is reversed with MCT1 inhibition. 3IL has been synthesised and purified. Ultrasound labile liposomes with an average hydrodynamic diameter of 120 nm have been synthesised and encapsulation of 3 BP has been confirmed and tested with cells with and without ultrasound. Conclusion Cell toxicity of halogenated lactate/pyruvate analogues has been confirmed. A novel iodinated analogue has been synthesised. These molecules have been encapsulated within ultrasound labile liposomes. A novel drug delivery system has been engineered for the delivery of metabolic inhibitors targeting the lactate/pyruvate transporter (MCT1) for targeted cancer therapy.

INDEL variation in the regulatory region of the major flowering time gene LanFTc1 is associated with vernalization response and flowering time in narrow-leafed lupin (Lupinus angustifolius L.).

Narrow‐leafed lupin (Lupinus angustifolius L.) cultivation was transformed by 2 dominant vernalization‐insensitive, early flowering time loci known as Ku and Julius (Jul), which allowed expansion into shorter season environments. However, reliance on these loci has limited genetic and phenotypic diversity for environmental adaptation in cultivated lupin. We recently predicted that a 1,423‐bp deletion in the cis‐regulatory region of LanFTc1, a FLOWERING LOCUS T (FT) homologue, derepressed expression of LanFTc1 and was the underlying cause of the Ku phenotype. Here, we surveyed diverse germplasm for LanFTc1 cis‐regulatory variation and identified 2 further deletions of 1,208 and 5,162 bp in the 5' regulatory region, which overlap the 1,423‐bp deletion. Additionally, we confirmed that no other polymorphisms were perfectly associated with vernalization responsiveness. Phenotyping and gene expression analyses revealed that Jul accessions possessed the 5,162‐bp deletion and that the Jul and Ku deletions were equally capable of removing vernalization requirement and up‐regulating gene expression. The 1,208‐bp deletion was associated with intermediate phenology, vernalization responsiveness, and gene expression and therefore may be useful for expanding agronomic adaptation of lupin. This insertion/deletion series may also help resolve how the vernalization response is mediated at the molecular level in legumes.

Behavioral and Emotional Problems in Gender-Nonconforming Children: A Canadian Community-Based Study

To examine childhood gender nonconformity (GNC) and psychological well-being in a community-based sample using measures that bridge clinical and nonclinical literature. Caregivers reported on the GNC (Gender Identity Questionnaire for Children [GIQC]) and behavioral and emotional problems (Child Behavior Checklist [CBCL]) of their children aged 6 to 12 years (N= 1719, 48.8% boys). The GIQC was compared to the commonly used single-item proxy, CBCL Item 110 ("wishes to be of the opposite sex"). Using the GIQC, 2.3% of boys and 2.8% of girls showed GNC levels comparable to those of children referred clinically for gender dysphoria (GD). Item 110 was endorsed for 1.7% of boys and 1.8% of girls. These measures corresponded, but Item 110 endorsement was biased toward more extreme GNC. Among boys, increased GNC on the GIQC, but not Item 110, corresponded with increased clinical-range CBCL problems. Among girls, Item 110 endorsement was associated with increased clinical-range Externalizing problems, whereas the GIQC indicated that intermediate gender expression was associated with fewer externalizing problems. Overall, rates of clinical-range CBCL problems among GNC children were consistent with those reported for GD-referred children. The scope of mental health risk among community children who exhibit GNC is likely considerably greater than previously recognized. A substantial minority of community children show GNC and mental health risk levels comparable to those seen among GD-referred children. Also, compared to the GIQC, a more comprehensive GNC measure, CBCL Item 110 is likely useful only for detecting extreme manifestations of GNC, which may affect associations with mental health.

Fine-tuning of FOXO3A in cHL as a survival mechanism and a hallmark of abortive plasma cell differentiation

AbstractWe recently found that FOXO1 repression contributes to the oncogenic program of classical Hodgkin lymphoma (cHL). Interestingly, FOXO3A, another member of the FOXO family, was reported to be expressed in the malignant Hodgkin and Reed-Sternberg cells of cHL at higher levels than in non-Hodgkin lymphoma subtypes. We thus aimed to investigate mechanisms responsible for the maintenance of FOXO3A as well as the potential role of FOXO3A in cHL. Here, we show that high FOXO3A levels in cHL reflect a B-cell-differentiation–specific pattern. In B cells, FOXO3A expression increases during the process of centroblast to plasma cell (PC) differentiation. FOXO3A levels in cHL were found higher than in germinal center B cells, but lower than in terminally differentiated PCs. This intermediate FOXO3A expression in cHL might manifest the “abortive PC differentiation” phenotype. This assumption was further corroborated by the finding that overexpression of FOXO3A in cHL cell lines induced activation of the master PC transcription factor PRDM1α. As factors attenuating FOXO3A expression in cHL, we identified MIR155 and constitutive activation of extracellular signal-regulated kinase. Finally, we demonstrate the importance of FOXO3A expression in cHL using an RNA interference approach. We conclude that tightly regulated expression of FOXO3A contributes to the oncogenic program and to the specific phenotype of cHL.

β2 Integrins Regulate Migratory Properties of M1 and M2 Macrophages during Inflammation

BackgroundThe development of chronic inflammation depends on the balance between accumulation of pro‐inflammatory, classically activated (M1) and alternatively activated (M2) macrophages within the injured tissue. The mesenchymal migration and accumulation of macrophages in the inflamed sites largely depend on myeloid‐specific αMβ2 and αDβ2 integrins, which share similar ligands but have different densities on different subsets of macrophages. The level of integrin expression regulates cell migration. Namely, a low‐intermediate expression supports cell motility, while a high expression generates strong adhesion that prevents cell migration. In this project, we tested how αMβ2 and αDβ2 integrins are involved in the migration and retention of M1 and M2 macrophages at the site of inflammation.Method and ResultsTo test our hypothesis, wild type (WT), αD−/− and αM−/− mice were intraperitoneally injected with thioglycollate to generate macrophages. Isolated macrophages were treated with IFN‐γ and IL‐4 for 4 days to activate M1 and M2 phenotypes, respectively. Integrin levels on macrophages were evaluated by qPCR and FACS. We found that the expression of αD was strongly upregulated on M1 macrophages, but not on M2 macrophages, while the level of αM remained unchanged on both subsets. To test the effect of integrins on the migration of M1 and M2 macrophages, we labeled cells with red and green fluorescent dyes and applied in vitro migration assay in 3D fibrin matrix.Our results showed that WT M2 macrophages possess significantly stronger migratory properties compared to WT M1 macrophages. To understand the contribution of individual integrins we used αD−/− and αM−/− macrophages. The results demonstrated that while αM‐deficiency had no significant effect, αD‐deficiency improved the migration of M1 macrophages. In contrast, both αD‐ and αM‐deficiency reduced the migration of M2 macrophages. To verify these results, we tested in vivo macrophage migration using the model of resolution of peritoneal inflammation. M1 and M2 macrophages, labeled with red and green fluorescent dyes respectively, were injected into the peritoneal cavity of mice with induced peritoneal inflammation. After 3 days, we evaluated the ability of macrophages to emigrate from the peritoneal cavity to lymphatics. Our results corresponded to our in vitro migratory assay. M2 macrophages emigrate significantly faster, αD‐deficiency improves the efflux of M1 macrophages and αD‐ and αM‐deficiency reduced the emigration of M2 macrophages.ConclusionTaken together, these data suggest that integrin αM, which has an intermediate expression on M1 and M2 macrophages, supports macrophage migration. In contrast, integrin αD, which has a high expression on M1 macrophages, prevents the migration of these cells and can promote the retention of M1 macrophages in the inflamed sites. Therefore, integrin αD is a potential therapeutic target to prevent pro‐inflammatory macrophage accumulation and development of chronic inflammation.Support or Funding InformationThis project is funded by NIH and the project number is 5R01DK102020‐05This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MP03-04 BRAF V600E MUTATION PROMOTED EXCESS OF HORMONE AND TUMORIGENESIS IN ADRENAL CORTICAL ADENOMA WITH AUTONOMOUS CORTISOL SECRETION

You have accessJournal of UrologyAdrenal1 Apr 2018MP03-04 BRAF V600E MUTATION PROMOTED EXCESS OF HORMONE AND TUMORIGENESIS IN ADRENAL CORTICAL ADENOMA WITH AUTONOMOUS CORTISOL SECRETION Kazuyuki Numakura, Taketoshi Nara, Sohei Kanda, Hiroshi Tsuruta, Mitsuru Saito, Shintaro Narita, Takamitsu Inoue, Shigeru Satoh, and Tomonori Habuchi Kazuyuki NumakuraKazuyuki Numakura More articles by this author , Taketoshi NaraTaketoshi Nara More articles by this author , Sohei KandaSohei Kanda More articles by this author , Hiroshi TsurutaHiroshi Tsuruta More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Shigeru SatohShigeru Satoh More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.3047AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The incidence of incidental adrenal tumors is increasing because of recent screening improvements in imaging modalities. A surgery is performed not only for functional adrenal tumors but also for suspected malignancy or growing size in preoperatively diagnosed non-functioning cortical adenoma (NCA). However, preoperative diagnosis of NCA is often inaccurate. BRAF was well known as tumorigenic driver gene in bowel intestine and other organs. However, an implication of BRAF in adrenal cortical adenoma has never investigated definitely. The purpose of this study was to analyze a clinical meaning of BRAF V600E mutation for acquiring endocrinological activity and tumorigenesis in adrenal cortical adenomas. METHODS In Akita University from September 1997 to July 2017, we performed 80 laparoscopic adrenalectomies for Cushing′s syndrome (CS), subclinical Cushing′s syndrome (SCS) and NCA. The diagnosis of them were determined on the basis of preoperative endocrinological evaluations. We analyzed expression of total BRAF and BRAF V600E mutation in adrenal cortical adenoma patients (CS in 15, SCS in 15, and NCA in 10) using by immunohistochemistry (IHC). RESULTS The correlation observed between expression of total BRAF and BRAF V600E mutation was very strong (r = 0.869). BRAF expression was high in 13 (26%), intermediate in 10 (20%), and low in 17 (34%) (Figure 1). High and intermediate expression of BRAF V600E were detected more frequently in CS (13/15 [86.7%], p<0.001) and SCS (10/15 [66.7%], p=0.003) compared with NCA (0/10 [0%]). No clinical factor, such as tumor diameter, endocrinological factors, and comorbidities, was different in groups. CONCLUSIONS BRAF V600E mutation might be a driver mutation to promote inappropriate cortisol secretion and be a target molecular in adrenal cortical adenoma with autonomous cortisol secretion. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e22 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Kazuyuki Numakura More articles by this author Taketoshi Nara More articles by this author Sohei Kanda More articles by this author Hiroshi Tsuruta More articles by this author Mitsuru Saito More articles by this author Shintaro Narita More articles by this author Takamitsu Inoue More articles by this author Shigeru Satoh More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Induction and Maintenance of CX3CR1-Intermediate Peripheral Memory CD8+ T Cells by Persistent Viruses and Vaccines

SummaryThe induction and maintenance of T cell memory is critical to the success of vaccines. A recently described subset of memory CD8+ T cells defined by intermediate expression of the chemokine receptor CX3CR1 was shown to have self-renewal, proliferative, and tissue-surveillance properties relevant to vaccine-induced memory. We tracked these cells when memory is sustained at high levels: memory inflation induced by cytomegalovirus (CMV) and adenovirus-vectored vaccines. In mice, both CMV and vaccine-induced inflationary T cells showed sustained high levels of CX3R1int cells exhibiting an effector-memory phenotype, characteristic of inflationary pools, in early memory. In humans, CX3CR1int CD8+ T cells were strongly induced following adenovirus-vectored vaccination for hepatitis C virus (HCV) (ChAd3-NSmut) and during natural CMV infection and were associated with a memory phenotype similar to that in mice. These data indicate that CX3CR1int cells form an important component of the memory pool in response to persistent viruses and vaccines in both mice and humans.

Intermediate filament protein expression pattern and inflammatory response changes in kidneys of rats receiving doxorubicin chemotherapy and quercetin

The aim of this study was to explore the potential effects of quercetin (QUR) on doxorubicin (DOX)-induced nephrotoxicity. Fifty male rats were assigned to five groups (10 rats each): a control group, a DOX-treated group (total dose, 15 mg/kg bw, intraperitoneally), a QUR-treated group (50 mg/kg bw/day, orally), a prophylaxis co-treated group, and a therapeutic co-treated group. Biochemical parameters and renal function were measured. Moreover, kidney tissues were homogenized for inflammatory marker evaluation and real-time qPCR analysis to determine the changes in intermediate filament protein mRNA levels (desmin, vimentin, connexin 43 and nestin). QUR exhibited a significant nephroprotective effect, particularly when it was administered prior to and simultaneously with DOX treatment (prophylaxis co-treated group). This role was biochemically demonstrated by the significant modulation of DOX-induced body weight loss, hypoproteinemia, and elevated serum creatinine and urea. Moreover, QUR attenuated the inflammatory response as shown by decreased renal nitric oxide, tumor necrosis factor-α production and myeloperoxidase activity elicited by DOX injection. These biochemical improvements were accompanied by a significant histopathological restoration of rat kidney tissue and successful down-regulation of the intermediate filament protein mRNA levels, indicating amelioration of DOX-induced podocyte injury. Taken together, these results conclusively demonstrated that QUR administration has a prophylactic effect on DOX-induced injury in the rat kidney.

Mycoplasma bovis-Induced Inhibition of Bovine Peripheral Blood Mononuclear Cell Proliferation Is Ameliorated after Blocking the Immune-Inhibitory Programmed Death 1 Receptor.

Mycoplasma bovis-induced immune suppression is a major obstacle faced by the host for controlling infections. M. bovis impairment of antigen-specific T-cell responses is achieved through inhibiting the proliferation of peripheral blood mononuclear cells (PBMCs). This impairment may contribute to the persistence of M. bovis infection in various sites, including lungs, and its systemic spread to various organs such as joints, with the underlying mechanisms remaining elusive. Here, we elucidated the role of the immune-inhibitory receptor programmed death 1 (PD-1) and its ligand (PD-L1) in M. bovis infection. Flow cytometry (FCM) analyses revealed an upregulation of PD-L1 expression on tracheal and lung epithelial cell lines after M. bovis infection. In addition, we found increased PD-L1 expression on purified lung lavage macrophages following M. bovis infection by FCM and determined its localization by immunofluorescence analysis comparing infected and control lung tissue sections. Moreover, M. bovis infection increased the expression of the PD-1 receptor on total PBMCs and in gated CD4+ and CD8+ T-cell subpopulations. We demonstrated that M. bovis infection induced a significant decrease in CD4+ PD-1INT and CD8+ PD-1INT subsets with intermediate PD-1 expression, which functioned as progenitor pools giving rise to CD4+ PD-1HIGH and CD8+ PD-1HIGH subsets with high PD-1 expression levels. We blocked PD-1 receptors on PBMCs using anti-PD-1 antibody at the beginning of infection, leading to a significant restoration of the proliferation of PBMCs. Taken together, our data indicate a significant involvement of the PD-1/PD-L1 inhibitory pathway during M. bovis infection and its associated immune exhaustion, culminating in impaired host immune responses.

Abstract P1-07-35: Adipokine receptor CAP1 expression as predictor of survival outcome among breast cancer patients

Abstract Background: Obesity is a challenging health concern in breast cancer. Not only are obese women at higher risk of breast cancer, but if diagnosed with the disease, obesity is also impairing the prognosis of a breast cancer. Understanding the underlying biology behind these associations are key to improve survival rates in breast cancer. In obesity, the more abundant, activated adipose tissue may lead to insulin resistance and secretion of soluble mediators that may contribute to impaired breast cancer outcomes observed among obese women. The adipokine resistin is implied to be an important link between obesity and insulin resistance, and to be elevated among breast cancer patients. Yet, the expression of the newly identified resistin receptor CAP1 and its impact on breast cancer prognosis is poorly understood. Purpose: To evaluate the impact by the adipokine receptor CAP1 tumor expression on breast cancer outcomes. Experimental Design: CAP1 mRNA expression was explored among 1,881 primary breast tumors. Associations between CAP1 expression, categorized into tertiles, and survival outcomes were estimated using the Kaplan-Meier method and log-rank test, and by multivariable Cox regression models providing adjusted hazard ratios (HRadj) with 95% confidence intervals (CI). Independent biological network interaction analyzes were conducted in a subset of 1,105 invasive breast carcinomas within The Cancer Genome Atlas (TCGA) project. Results: CAP1 was linked to poor tumor characteristics with higher CAP1 expression found among estrogen receptor (ER)-negative tumors, relative to ER-positive tumors, as well as with increasing tumor grade (P=0.025). High CAP1 tumor expression was further associated with shorter overall survival (HRadj 1.54; 95% CI, 1.11-2.13) and relapse-free survival (HRadj 1.47; 95% CI, 1.10-1.96), compared with low or intermediate CAP1 expression, particularly among ER-positive tumors or lymph node positive tumors. Additionally, CAP1 was identified in complex with genes involved in growth promoting and cell motility processes. Conclusion: These results highlight the potential role of the resistin receptor CAP1 regarding breast cancer outcomes. Further investigations are needed to elucidate the biological and clinical implication of the resistin-CAP1 link in obesity-associated breast cancer. Citation Format: Rosendahl AH, Bergqvist M, Kimbung S, Borgquist S. Adipokine receptor CAP1 expression as predictor of survival outcome among breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-35.

Leptin is a dose-dependent marker of caloric restriction in adipose tissues located in different parts of the mouse body

We investigated leptin expression in four fat tissues located in different body parts of male mice. In the absence of caloric restriction (CR), the highest relative levels of leptin mRNA were detected in epididymal fat (EF). Lateral abdominal fat (LAF) and subcutaneous fat (SF) had intermediate leptin expression levels, while the lowest leptin mRNA content was revealed in brown adipose tissue (BAT). We also investigated differential regulation of leptin expression in these adipose tissues following diets that were based on various levels of CR for 10 weeks. The largest reduction of leptin mRNA levels by CR was observed in adipose tissue where maximal leptin mRNA expression was detected in the absence of CR. Leptin protein expression was also decreased by CR as revealed by immunohistochemistry. The changes in leptin mRNA and protein expression positively correlated with adipocyte size that was also diminished with the increase in the strength of CR.

Induction and maintenance of CX3CR1-intermediate peripheral memory CD8 T cells by persistent viruses and novel vaccines

The induction and maintenance of T-cell memory is critical to the success of novel vaccines. A newlydescribed subset of memory CD8 T-cells defined by intermediate expression of the chemokine receptor CX3CR1, were shown to have self-renewal, proliferative and tissue-surveillance properties relevant to vaccine-induced memory. We tracked these cells in situations where memory is sustained at high levels – memory “inflation” induced by cytomegalovirus (CMV) and adenovirus-vectored vaccines. In mice, both CMV and vaccine-induced inflationary T-cells showed sustained high levels of CX3R1int cells exhibiting an effector-memory phenotype, characteristic of inflationary pools. In humans, CX3CR1int CD8 T-cells were strongly induced following administration of an adenovirus-vectored vaccine for HCV (ChAd3-NSmut) and during natural CMV infection, and were associated with a memory phenotype similar to that in mice. These data indicate that CX3CR1int memory cells form a substantial component of the memory pool in response to persistent viruses and vaccines in both mouse and man.

Estimating and testing direct genetic effects in directed acyclic graphs using estimating equations.

ABSTRACTIn genetic association studies, it is important to distinguish direct and indirect genetic effects in order to build truly functional models. For this purpose, we consider a directed acyclic graph setting with genetic variants, primary and intermediate phenotypes, and confounding factors. In order to make valid statistical inference on direct genetic effects on the primary phenotype, it is necessary to consider all potential effects in the graph, and we propose to use the estimating equations method with robust Huber–White sandwich standard errors. We evaluate the proposed causal inference based on estimating equations (CIEE) method and compare it with traditional multiple regression methods, the structural equation modeling method, and sequential G‐estimation methods through a simulation study for the analysis of (completely observed) quantitative traits and time‐to‐event traits subject to censoring as primary phenotypes. The results show that CIEE provides valid estimators and inference by successfully removing the effect of intermediate phenotypes from the primary phenotype and is robust against measured and unmeasured confounding of the indirect effect through observed factors. All other methods except the sequential G‐estimation method for quantitative traits fail in some scenarios where their test statistics yield inflated type I errors. In the analysis of the Genetic Analysis Workshop 19 dataset, we estimate and test genetic effects on blood pressure accounting for intermediate gene expression phenotypes. The results show that CIEE can identify genetic variants that would be missed by traditional regression analyses. CIEE is computationally fast, widely applicable to different fields, and available as an R package.

Predictive value of BRCA1/2 mRNA expression for response to neoadjuvant chemotherapy in BRCA-negative breast cancers.

It is well known that BRCA1 and BRCA2 play a central role in DNA repair, but the relationship between BRCA1 and BRCA2 mRNA expression and response to neoadjuvant chemotherapy in sporadic breast cancer patients has not been well established. Here, we investigate the association between BRCA1 or BRCA2 mRNA expression levels and pathological response in 674 BRCA1/2 mutation‐negative breast cancer patients who received neoadjuvant chemotherapy. BRCA1 and BRCA2 mRNA expression were assessed using quantitative real‐time polymerase chain reaction in core biopsy breast cancer tissue obtained prior to the initiation of neoadjuvant chemotherapy. A total 129 patients (19.1%) achieved pathological complete response (pCR) after neoadjuvant chemotherapy. Among patients treated with anthracycline‐based chemotherapy (n = 531), BRCA1 mRNA low expression patients had a significantly higher pCR rate than intermediate or high BRCA1 mRNA expression groups (24.6% vs 16.8% or 14.0%, P = .031) and retained borderline significance (OR = 1.54, 95% CI = 0.93‐2.56, P = .094) in multivariate analysis. Among the 129 patients who received a taxane‐based regimen, pCR rate showed no differences in BRCA1 low, intermediate, and high mRNA level subgroups (19.6%, 26.8% and 21.4%, respectively; P = .71). BRCA2 mRNA level was not associated with pCR rate in the anthracyline‐based treated subgroup (P = .60) or the taxane‐based regimen subgroup (P = .82). Taken together, our findings suggested that BRCA1 mRNA expression could be used as a predictive marker in BRCA1/2 mutation‐negative breast cancer patients who received neoadjuvant anthracycline‐based treatment.

An Effective Model of the Retinoic Acid Induced HL-60 Differentiation Program

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.

An intermediate level of CD161 expression defines a novel activated, inflammatory, and pathogenic subset of CD8+ T cells involved in multiple sclerosis

Several lines of evidence support a key role for CD8+ T cells in central nervous system tissue damage of patients with multiple sclerosis. However, the precise phenotype of the circulating CD8+ T cells that may be recruited from the peripheral blood to invade the CNS remains largely undefined to date. It has been suggested that IL-17 secreting CD8 (Tc17) T cells may be involved, and in humans these cells are characterized by the expression of CD161. We focused our study on a unique and recently described subset of CD8 T cells characterized by an intermediate expression of CD161 as its role in neuroinflammation has not been investigated to date. The frequency, phenotype, and function of CD8+ T cells with an intermediate CD161 expression level were characterized ex-vivo, in vitro, and in situ using RNAseq, RT-PCR, flow cytometry, TCR sequencing, and immunohistofluorescence of cells derived from healthy volunteers (n = 61), MS subjects (n = 90), as well as inflammatory (n = 15) and non-inflammatory controls (n = 6). We report here that CD8+CD161int T cells present characteristics of effector cells, up-regulate cell-adhesion molecules and have an increased ability to cross the blood-brain barrier and to secrete IL-17, IFNγ, GM-CSF, and IL-22. We further demonstrate that these cells are recruited and enriched in the CNS of MS subjects where they produce IL-17. In the peripheral blood, RNAseq, RT-PCR, high-throughput TCR repertoire analyses, and flow cytometry confirmed an increased effector and transmigration pattern of these cells in MS patients, with the presence of supernumerary clones compared to healthy controls. Our data demonstrate that intermediate levels of CD161 expression identifies activated and effector CD8+ T cells with pathogenic properties that are recruited to MS lesions. This suggests that CD161 may represent a biomarker and a valid target for the treatment of neuroinflammation.

Inhibition of Aurora Kinase A Synergistically Enhances Cytotoxicity in Ovarian Clear Cell Carcinoma Cell Lines Induced by Cisplatin: A Potential Treatment Strategy.

This study aims to clarify the incidence of Aurora kinase A (Aurora-A) protein expression and its correlation with clinical parameters in ovarian clear cell carcinoma (OCCC) tumor tissues. In addition, we assessed the efficacy of ENMD-2076, a novel selective Aurora-A inhibitor, in combination with chemotherapeutic agents for the treatment of OCCC. Aurora-A protein expression was determined by immunohistochemical staining of OCCC specimens from 56 patients to evaluate its correlation with clinical outcomes in OCCC. In the in vitro study, 6 OCCC cell lines were exposed to ENMD-2076 in combination with cisplatin, SN38, doxorubicin, or paclitaxel, and cell proliferation, cell cycle distribution, and apoptosis were assessed. The 5-year survival rates of International Federation of Gynecology and Obstetrics stages IC3 to IV patients with intermediate or strong Aurora-A expression were significantly lower than those of patients with negative or weak Aurora-A expression. Increased Aurora-A expression was associated with significantly worse overall survival of International Federation of Gynecology and Obstetrics stages IC3 to IV patients (21% vs 77%). Multivariate analysis revealed that Aurora-A expression was an independent prognostic factor for stages IC3 to IV OCCC patients. Furthermore, synergistic effects were observed with ENMD-2076 in combination with cisplatin or SN-38 in 4 of the 6 tested cell lines. ENMD-2076 dramatically enhanced apoptosis and cell cycle arrest at the G2/M phase induced by cisplatin. Aurora-A is a promising biomarker that is predictive of patient outcomes and a potential target for OCCC. The results suggested that chemotherapy, including ENMD-2076 in combination with cisplatin, is a potential treatment modality for patients with OCCC.