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The validity and reliability of research on the nature and extent of sexual assault tends to be affected by different definitions, methodologies, and measurements. As a result, two important aspects of sexual assault associated with patterns of symptom expression and therapeutic interventions are not often reflected in the research: the severity of the assault, including the duration of the abuse, and the age at the time of the assault and the gender of the victim. This research is based on intake forms from Hawai'i's only statewide provider of services to the victims of sexual assault. The analyses reveal that significant differences exist between male and female victims, by age and by assault characteristics, including the type of sexual assault, use of force and injury, length of assault, and the relationship between victim and offender. [ABSTRACT FROM AUTHOR]

Detection of extranodal extension on histopathology in surgically treated head and neck squamous cell carcinoma indicates poor prognosis. However, there is no consensus on the diagnostic criteria, interpretation, and reporting of histology detected extranodal extension, which has contributed to conflicting evidence in the literature, and likely clinical inconsistency. The Head and Neck Cancer International Group conducted a three-round modified Delphi process with a group of 19 international pathology experts representing 15 national clinical research groups to generate consensus recommendations for histology detected extranodal extension diagnostic criteria. The expert panel strongly agreed on terminology and diagnostic features for histology detected extranodal extension and soft tissue metastasis. Moreover, the panel reached consensus on reporting of histology detected extranodal extension and on nodal sampling. These consensus recommendations, endorsed by 19 organisations representing 34 countries, are a crucial development towards standardised diagnosis and reporting of histology detected extranodal extension, and more accurate data collection and analysis., Competing Interests: Declaration of interests HM is the Director and a shareholder of Warwickshire Head and Neck Clinic; chair of the Head and Neck Cancer International Group; past president of the British Association of Head and Neck Oncologists; reports receiving honoraria from AstraZeneca; is on the Speaker's Bureau for Merck Sharpe Dohme (MSD), Sanofi Pasteur, and Merck; received funding from GSK Biologicals, MSD, Sanofi Pasteur, GSK, and AstraZeneca; and received travel accommodation expenses from Sanofi Pasteur, MSD, and Merck. WML is the chair of American Joint Committee on Cancer 9th Version head and neck task force. RDC is a member of a steering committee for a phase 3 clinical trial of neoadjuvant pembrolizumab sponsored by Merck and also has a non-financial relationship with Caris Life Sciences as a member of their Precision Oncology Alliance. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Cancer/testis antigens (CTAs) are widely expressed in melanoma and lung cancer, emerging as promising targets for vaccination strategies and T-cell-based therapies in these malignancies. Despite recognizing the essential impact of intratumoral heterogeneity on clinical responses to immunotherapy, our understanding of intratumoral heterogeneity in CTA expression has remained limited. We employed single-cell mRNA sequencing to delineate the CTA expression profiles of cancer cells in clinically derived melanoma and lung cancer samples. Our findings reveal a high degree of intratumoral transcriptional heterogeneity in CTA expression. In melanoma, every cell expressed at least one CTA. However, most individual CTAs, including the widely used therapeutic targets NY-ESO-1 and MAGE, were confined to subpopulations of cells and were uncoordinated in their expression, resulting in mosaics of cancer cells with diverse CTA profiles. Coordinated expression was observed, however, mainly among highly structurally and evolutionarily related CTA genes. Importantly, a minor subset of CTAs, including PRAME and several members of the GAGE and MAGE-A families, were homogenously expressed in melanomas, highlighting their potential as therapeutic targets. Extensive heterogeneity in CTA expression was also observed in lung cancer. However, the frequency of CTA-positive cancer cells was notably lower and homogenously expressed CTAs were only identified in one of five tumors in this cancer type. Our findings underscore the need for careful CTA target selection in immunotherapy development and clinical testing and offer a rational framework for identifying the most promising candidates., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies., Competing Interests: Declaration of interests E.J.S. is a co-founder, scientific advisor, and equity holder of Intellia Therapeutics and a member of the scientific advisory board of Tessera Therapeutics. J.G.D. consults for Microsoft Research, BioNTech, Servier, and Pfizer. J.G.D. consults for and has equity in Tango Therapeutics. J.G.D. serves as a paid scientific advisor to the Laboratory for Genomics Research, funded in part by GSK, and the Innovative Genomics Institute, funded in part by Apple Tree Partners. J.G.D. receives funding support from the Functional Genomics Consortium: Abbvie, Bristol Myers Squibb, Janssen, and Merck. J.G.D.’s interests are reviewed and managed by the Broad Institute in accordance with its conflict of interest policies., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Extranodal extension (ENE) in lymph node metastases is one of the most important prognostic factors in head and neck squamous cell carcinomas. Studies have shown inconsistency among pathologists in the assessment of ENE. The aims of this study were: (1) to determine the interrater and intrarater reliability and agreement in the assessment of ENE among Danish pathologists and (2) to test if a standardized assessment method may increase interrater agreement., Methods: Four Danish head and neck pathologists assessed ENE presence or absence in 120 histological slides from lymph nodes with oropharyngeal squamous cell carcinoma metastases (first round). Subsequently, guidelines were introduced to the pathologists and a new assessment was performed (second round). Finally, two of the pathologists assessed the slides to determine intrarater reliability and agreement (third round)., Results: Interrater kappa coefficients varied between 0.57 and 0.67 in the first round and between 0.59 and 0.72 in the second round. The intrarater agreement between round 2 and 3 was 0.88 for pathologist 1 and 0.92 for pathologist 2 with resulting kappa coefficients of 0.76 (95% CI 0.64-0.88) and 0.84 (95% CI 0.74-0.94), respectively., Conclusion: We found a moderate level of reliability and agreement among pathologists for ENE in lymph node metastases from oropharyngeal squamous cell carcinomas. The intrarater reliability and agreement was generally higher than interrater measures. Interrater agreement was slightly improved by standardized assessment., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Poor oral health is common in the United States; however, it is much more common in African Americans, Hispanics, and other racial/ethnic minorities. Almost one in five low-income adults states that their mouth and teeth are in poor condition. Twenty-nine percent of Americans have no dental insurance. Patients who have active infections are at greater risk for prosthetic joint infection. Optimization in these vulnerable groups should focus on treating active infections, with a prioritization of free clinics, academic clinics, and websites, such as "The Neighborhood Navigator," and easily accessible surgical consults., (Copyright © 2021 by the American Academy of Orthopaedic Surgeons.)

Digitalisation of pathology slides allows pathologists to make diagnoses using a high-resolution computer screen instead of a conventional microscope. In 2020/21, the four pathology departments in the Region of Southern Denmark implemented digital pathology for all histologic samples. Going digital necessitated optimisation of workflows and training of pathologists, avoiding a reduction in diagnostic quality. This review describes the process for realisation of digital pathology and its future perspectives, including artificial intelligence algorithms to be implemented.

Background: Completion of Medicare Annual Wellness Visits (AWV) and documentation of Hierarchical Condition Categories (HCC) are important metrics in accountable care organizations (ACO) with quality and financial implications. To improve performance in large healthcare organizations, quality improvement (QI) efforts need to be scaled up in a way that is feasible within available system-wide resources., Objective: We describe a 3-year effort using a multifaceted QI framework called the fractal management system for AWV and HCC performance., Design: Pre-post evaluation of a multi-level, health system-wide QI management system intervention between 2018 and 2020. The system provided project management, coaching, communications, feedback of performance, and health informatics., Participants: The intervention was delivered to all 97 primary care practices within an Ohio-based accountable care organization, comprising 72,603 attributed Medicare and Medicare Advantage patients as of 2018. Eighty-nine of these practices were included in the analysis., Approach: AWV completion was defined as percent of eligible patients with a documented AWV during the calendar year. HCC completion was defined as documented reassessment of all prior-year HCC conditions., Key Results: AWV completion at the practice level increased from 23.7% (SD .14) in 2018 to 34.9% (SD .18) in 2019, and 59.8% (SD .17) in 2020. This was a statistically significant effect of time on AWV completion rates overall (F[2, 87] = 164.43, p < .000). More than half (56.2%) of practices met or exceeded the 60% goal in 2020. Practice-level HCC completion tracking started in 2019 (M = 75.9%, SD 7.4%) and increased in 2020 (M = 79.7%, SD 7.1%); t(172) = 2.0, p < .001., Conclusions: AWV and HCC performance goals were met in 2020, despite service disruptions due to COVID-19. The QI approach we used is applicable to other problems and other large healthcare systems., (© 2022. The Author(s) under exclusive licence to Society of General Internal Medicine.)

Purpose: The purpose of this study was to determine the effects of blood flow restriction (BFR) using a pneumatic tourniquet on CD34 + cells, platelets, white blood cells, neutrophils, lymphocytes, lactate, and glucose compared with standard exercise., Methods: Fifteen healthy volunteers (8 males and 7 females, 28.6 ± 3.6 years old) who were able to perform the exercise sessions on a VersaClimber participated. Participants were randomized to undergo an experimental (EXP) occluded testing session using the pneumatic tourniquets on all 4 extremities and a control (CON) session. The exercise protocol concluded after 9 minutes or when participants reached a rating of perceived exertion of 20. Blood draws were performed before testing and immediately after the exercise session. Blood analysis consisted of complete blood counts as well as flow cytometry to measure peripheral CD34 + counts as a marker for hematopoietic progenitor cells (HPCs)., Results: A significant increase from before to after exercise values was observed in both the EXP and CON groups with CD34 + , WBC counts, platelets, and lymphocytes; however, no differences existed between EXP and CON groups for any variable. CD34 + increased in the EXP (3.1 ± 1.6 vs. 4.3 ± 1.8 cells · L -1 ; P < .001) and CON (3.3 ± 1.9 vs. 4.4 ± 1.4 cells · L -1 ; P < .001) sessions. White blood cells also significantly increased in both the EXP (7.8 ± 1.4 vs. 11.8 ± 2.5 K · L -1 K · L -1 ; P < .001) and CON (7.5 ± 1.8 vs. 11.3 ± 3.0 K · L -1 ; P < .001) sessions. Platelets also increased in both the EXP (258.6 ± 52.5 vs. 309.9 ± 52.7 K · L -1 ; P < .001) and CON (263.1 ± 44.7 vs. 316.1 ± 43.9 K · L -1 ; P < .001) sessions, and conversely, a significant decrease in the average neutrophil counts in the EXP (mean difference = -13.7%; P < .001) and CON (mean difference = -13.2%; P < .001) sessions was observed. Lymphocyte counts in the EXP (mean difference = 22.8%; P < .001) and CON (mean difference = 19.3%; P < .001) sessions increased significantly., Conclusions: There were no significant differences in systemic cellular responses when undergoing aerobic-based exercise with and without a pneumatic tourniquet system., Level of Evidence: 2, prospective comparative study., (© 2022 The Authors.)

Epigenetic alteration has been proposed to give rise to numerous classic hallmarks of cancer. Impaired DNA methylation plays a central role in the onset and progression of several types of malignancies, and DNA methylation is mediated by DNA methyltransferases (DNMTs) consisting of DNMT1, DNMT3A, and DNMT3B. DNMTs are frequently implicated in the pathogenesis and aggressiveness of acute myeloid leukaemia (AML) patients. In this review, we describe and discuss the oncogenic roles of DNMT1, DNMT3A, and DNMT3B in AML. The clinical response predictive roles of DNMTs in clinical trials utilising hypomethylating agents (azacitidine and decitabine) in AML patients are presented. Novel hypomethylating agent (guadecitabine) and experimental DNMT inhibitors in AML are also discussed. In summary, hypermethylation of tumour suppressors mediated by DNMT1 or DNMT3B contributes to the progression and severity of AML (except MLL-AF9 and inv(16)(p13;q22) AML for DNMT3B), while mutation affecting DNMT3A represents an early genetic lesion in the pathogenesis of AML. In clinical trials of AML patients, expression of DNMTs is downregulated by hypomethylating agents while the clinical response predictive roles of DNMT biomarkers remain unresolved. Finally, nucleoside hypomethylating agents have continued to show enhanced responses in clinical trials of AML patients, and novel non-nucleoside DNMT inhibitors have demonstrated cytotoxicity against AML cells in pre-clinical settings., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The availability of multiple datasets comprising genome-scale RNAi viability screens in hundreds of diverse cancer cell lines presents new opportunities for understanding cancer vulnerabilities. Integrated analyses of these data to assess differential dependency across genes and cell lines are challenging due to confounding factors such as batch effects and variable screen quality, as well as difficulty assessing gene dependency on an absolute scale. To address these issues, we incorporated cell line screen-quality parameters and hierarchical Bayesian inference into DEMETER2, an analytical framework for analyzing RNAi screens ( https://depmap.org/R2-D2 ). This model substantially improves estimates of gene dependency across a range of performance measures, including identification of gold-standard essential genes and agreement with CRISPR/Cas9-based viability screens. It also allows us to integrate information across three large RNAi screening datasets, providing a unified resource representing the most extensive compilation of cancer cell line genetic dependencies to date.

DNMT1 is a target of approved anti-cancer drugs including decitabine. However, the prognostic value of DNMT1 protein expression in R-CHOP-treated diffuse large B-cell lymphomas (DLBCLs) remains unexplored. Here we showed that DNMT1 was expressed in the majority of DLBCL cases (n = 209/230, 90.9%) with higher expression in germinal centre B-cell-like (GCB)-DLBCL subtype. Low and negative DNMT1 expression (20% cut-off, n = 33/230, 14.3%) was predictive of worse overall survival (OS; p < 0.001) and progression-free survival (PFS; p < 0.001). Nonetheless, of the 209 DNMT1 positive patients, 33% and 42% did not achieve 5-year OS and PFS, respectively, indicating that DNMT1 positive patients showed considerably heterogeneous outcomes. Moreover, DNMT1 was frequently expressed in mitotic cells and significantly correlated with Ki-67 or BCL6 expression (r = 0.60 or 0.44, respectively; p < 0.001). We demonstrate that DNMT1 is predictive of DLBCL patients' survival, and suggest that DNMT1 could be a DLBCL therapeutic target due to its significant association with Ki-67., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Aims: Transient receptor potential channel melastatin 4 (TRPM4) is an ion channel that regulates influx of calcium cations (Ca 2+ ). Recent studies suggest that TRPM4 is an oncoprotein, and its up-regulated transcript level has been reported in diffuse large B cell lymphoma (DLBCL). We aimed to investigate TRPM4 protein expression pattern in non-malignant tissues and DLBCL cases, and its association with clinico-demographic parameters and survival in DLBCL., Methods and Results: Analysis of publicly available DLBCL microarray data sets showed that TRPM4 transcripts were up-regulated in DLBCL compared to normal germinal centre B (GCB) cells, were expressed more highly in the activated B cell-like DLBCL (ABC-DLBCL) subtype and higher TRPM4 transcripts conferred worse overall survival (OS) in R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL cases (P < 0.05). Our immunohistochemical analysis showed that TRPM4 was expressed in various human tissues but not in normal B cells within lymphoid tissues (reactive tonsil, lymph node and appendix). TRPM4 protein was present in 26% (n = 49 of 189) of our cohort of R-CHOP-treated DLBCL cases and this was associated significantly with more aggressive clinical parameters, including higher lactate dehydrogenase (LDH), Eastern Cooperative Oncology Group (ECOG) scores or stage (P < 0.01 for each of the parameters) and the ABC-DLBCL subtype (P = 0.016). TRPM4 positivity conferred significantly worse OS (P = 0.004) and progression-free survival (PFS) (P = 0.005). Worse OS remained associated significantly with TRPM4 positivity in multivariate analysis, including higher International Prognostic Index (IPI) or the non-GCB DLBCL phenotype (P < 0.05)., Conclusions: TRPM4 protein expression is up-regulated in DLBCL cases compared to non-malignant B cells with preferential expression in ABC-DLBCL cases, and it confers significantly poorer DLBCL patient outcomes., (© 2017 John Wiley & Sons Ltd.)

Extracellular membrane vesicles (EVs) function as vehicles of intercellular communication, but how the biomaterials they carry reach the target site in recipient cells is an open question. We report that subdomains of Rab7+ late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, where biomaterials associated with CD9+ EVs are delivered. EV-derived biomaterials were also found in the nuclei of host cells. The inhibition of nuclear import and export pathways abrogated the nuclear localization of EV-derived biomaterials or led to their accumulation therein, respectively, suggesting that their translocation is dependent on nuclear pores. Nuclear envelope invagination-associated late endosomes were observed in ex vivo biopsies in both breast carcinoma and associated stromal cells. The transcriptome of stromal cells exposed to cancer cell-derived CD9+ EVs revealed that the regulation of eleven genes, notably those involved in inflammation, relies on the nuclear translocation of EV-derived biomaterials. Our findings uncover a new cellular pathway used by EVs to reach nuclear compartment.

We present a multiplex analysis for genes known to have prognostic value in an attempt to design a clinically useful classification model in patients with diffuse large B-cell lymphoma (DLBCL). Real-time polymerase chain reaction was used to measure transcript levels of 28 relevant genes in 194 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). Including International Prognostic Index (IPI) as a variable in a penalized Cox regression, we investigated the association with disease progression for single genes or gene combinations in four models. The best model was validated in data from an online available R-CHOP treated cohort. With progression-free survival (PFS) as primary endpoint, the best performing IPI independent model incorporated the LMO2 and HLADQA1 as well as gene interactions for GCSAMxMIB1, GCSAMxCTGF and FOXP1xPDE4B. This model assigned 33% of patients (n = 60) to poor outcome with an estimated 3-year PFS of 40% vs. 87% for low risk (n = 61) and intermediate (n = 60) risk groups (P < 0·001). However, a simpler, IPI independent model incorporated LMO2 and BCL2 and assigned 33% of the patients with a 3-year PFS of 35% vs. 82% for low risk group (P < 0·001). We have documented the impact of a few single genes added to IPI for assignment in new drug trials., (© 2016 John Wiley & Sons Ltd.)

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL., Competing Interests: The authors declare no conflicts of interest.

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco-Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients.

Purpose of Review: Cancer cells utilize extracellular vesicles (EVs) as a means of transferring oncogenic proteins and nucleic acids to other cells to enhance the growth and spread of the tumor. There is an unexpected amount of similarities between these small, membrane-bound particles and enveloped virions, including protein content, physical characteristics (i.e., size and morphology), and mechanisms of entry and exit into target cells., Recent Findings: This review describes the attributes shared by both cancer-derived EVs, with an emphasis on breast cancer-derived EVs, and enveloped viral particles and discusses the methods by which virions can utilize the EV pathway as a means of transferring viral material and oncogenes to host cells. Additionally, the possible links between human papilloma virus and its influence on the miRNA content of breast cancer-derived EVs are examined., Summary: The rapidly growing field of EVs is allowing investigators from different disciplines to enter uncharted territory. The study of the emerging similarities between cancer-derived EVs and enveloped virions may lead to novel important scientific discoveries., Competing Interests: Conflicts of InterestToni M. Green, Mark F. Santos, Sanford H. Barsky, Germana Rappa, and Aurelio Lorico declare that they have no conflict of interest., (© Springer Science+Business Media New York 2016.)

Huntingtin-interacting protein 1-related (HIP1R) is an endocytic protein involved in receptor trafficking, including regulating cell surface expression of receptor tyrosine kinases. We have previously shown that low HIP1R protein expression was associated with poorer survival in diffuse large B-cell lymphoma (DLBCL) patients from Denmark treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In this multicenter study, we extend these findings and validate the prognostic and subtyping utility of HIP1R expression at both transcript and protein level. Using data mining on three independent transcriptomic datasets of DLBCL, HIP1R transcript was preferentially expressed in germinal center B-cell (GCB)-like DLBCL subtype (P<0.01 in all three datasets), and lower expression was correlated with worse overall survival (OS; P<0.01) and progression-free survival (PFS; P<0.05) in a microarray-profiled DLBCL dataset. At the protein level examined by immunohistochemistry, HIP1R expression at 30% cut-off was associated with GCB-DLBCL molecular subtype (P=0.0004; n=42), and predictive of OS (P=0.0006) and PFS (P=0.0230) in de novo DLBCL patients treated with R-CHOP (n=73). Cases with high FOXP1 and low HIP1R expression frequency (FOXP1(hi)/HIP1R(lo) phenotype) exhibited poorer OS (P=0.0038) and PFS (P=0.0134). Multivariate analysis showed that HIP1R<30% or FOXP1(hi)/HIP1R(lo) subgroup of patients exhibited inferior OS and PFS (P<0.05) independently of the International Prognostic Index. We conclude that HIP1R expression is strongly indicative of survival when utilized on its own or in combination with FOXP1, and the molecule is potentially applicable for subtyping of DLBCL cases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: TRIM21 (also known as Ro52) is an autoantigen in rheumatic disease and is predominantly expressed in leucocytes. Overexpression is associated with decreased proliferation, and the TRIM21 gene maps to a tumour suppressor locus. We therefore investigated the expression of TRIM21 in patients with diffuse large B-cell lymphoma (DLBCL) and its potential usefulness as a prognostic biomarker., Materials and Methods: TRIM21 expression levels were assessed by immunohistochemistry in lymphoma biopsies from three cohorts of patients with DLBCL: 42 patients with rheumatic disease treated with a cyclophosphamide, vincristine, doxorubicin and prednisone (CHOP)-like regimen, 76 CHOP-treated and 196 rituximab-CHOP-treated nonrheumatic patients. Expression was correlated with clinical and biomedical parameters. TRIM21 expression was assessed in relation to lymphocyte proliferation by quantitative PCR and correlated with (3) H-thymidine incorporation and propidium iodine staining., Results: TRIM21 expression levels differed in the lymphomas compared to normal lymphoid tissue, with reduced expression correlating with shorter overall survival in all three cohorts. In the two larger cohorts, progression-free survival was assessed and was also found to correlate with TRIM21 expression. The association was independent of commonly used clinical prognostic scores, lymphoma subtype and several previously reported prognostic biomarkers. In agreement with this clinical observation, we noted an inverse correlation between TRIM21 expression and proliferation of leucocytes in vitro., Conclusions: We show that loss of TRIM21 expression is associated with more aggressive lymphoma and increased proliferation, whereas maintenance of TRIM21 expression is associated with better prognosis in patients with DLBCL. Based on our findings, we suggest that TRIM21 should be considered as a novel biomarker for lymphoma characterization and for predicting patient survival., (© 2015 The Association for the Publication of the Journal of Internal Medicine.)

Interaction of breast cancer cells (BCCs) with stromal components is critical for tumor growth and metastasis. Here, we assessed the role of CD9 in adhesion, migration and invasiveness of BCCs. We used co-cultures of BCCs and bone marrow-derived multipotent mesenchymal stromal cells (MSCs), and analyzed their behavior and morphology by dynamic total internal reflection fluorescence, confocal and scanning electron microscopy. 83, 16 and 10% of contacts between MDA-MB-231 (MDA), MA-11 or MCF-7 cells and MSCs, respectively, resulted in MSC invasion. MDA cells developed long magnupodia, lamellipodia and dorsal microvilli, whereas long microvilli emerged from MA-11 cells. MCF-7 cells displayed large dorsal ruffles. CD9 knockdown and antibody blockage in MDA cells inhibited MSC invasion by 95 and 70%, respectively, suggesting that CD9 is required for this process. Remarkably, CD9-deficient MDA cells displayed significant alteration of their plasma membrane, harboring numerous peripheral and dorsal membrane ruffles instead of intact magnupodium/lamellipodium and microvillus, respectively. Such modification might explain the delayed adhesion, and hence MSC invasion. In agreement with this hypothesis, CD9-knockdown suppressed the metastatic capacity of MDA cells in mouse xenografts. Our data indicate that CD9 is implicated in BCC invasiveness and metastases by cellular mechanisms that involve specific CD9+ plasma membrane protrusions of BCCs.

The study of extracellular vesicles (EVs) in cancer progression is a complex and rapidly evolving field. Whole categories of cellular interactions in cancer which were originally presumed to be due solely to soluble secreted molecules have now evolved to include membrane-enclosed extracellular vesicles (EVs), which include both exosomes and shed microvesicles (MVs), and can contain many of the same molecules as those secreted in soluble form but many different molecules as well. EVs released by cancer cells can transfer mRNA, miRNA, and proteins to different recipient cells within the tumor microenvironment, in both an autocrine and paracrine manner, causing a significant impact on signaling pathways, mRNA transcription, and protein expression. The transfer of EVs to target cells, in turn, supports cancer growth, immunosuppression, and metastasis formation. This review focuses exclusively on breast cancer EVs with an emphasis on breast cancer-derived exosomes, keeping in mind that breast cancer-derived EVs share some common physical properties with EVs of other cancers.

Unlabelled: Tetraspanin-29 (CD9) is an integral membrane protein involved in several fundamental cell processes and in cancer metastasis. Here, characterization of a panel of breast cancer cells revealed a nuclear pool of CD9, not present in normal human mammary epithelial cells. Antibody binding to surface CD9 of breast cancer cells resulted in increased nuclear CD9 fluorescence. CD9 was also found, along with a plasma membrane-associated pool, in the nuclei of all primary ductal breast carcinoma patient specimens analyzed. In all patients, about 40% of the total CD9 cellular fluorescence was nuclear. CD9 colocalized at the nuclear level with CEP97, a protein implicated in centrosome function, and with the IGSF8, an established CD9 partner in the plasma membrane. Co-immunoprecipitation of CEP97 and IGSF8 with CD9 was shown in nuclear extracts from breast cancer cells expressing a CD9-GFP fusion protein. However, by fluorescence resonance energy transfer (FRET) analysis, no direct binding of CD9 with either protein was observed, suggesting that CD9 is part of a larger nuclear protein complex. CD9 depletion or exposure of parental breast cancer cells to anti-CD9 mAb resulted in polynucleation and multipolar mitoses. These data indicate that the nuclear CD9 pool has an important role in the mitotic process., Implications: The discovery of a nuclear pool of CD9 has prognostic and/or therapeutic potential for patients with ductal carcinoma of the breast., (©2014 American Association for Cancer Research.)

Using a genome-scale, lentivirally delivered shRNA library, we performed massively parallel pooled shRNA screens in 216 cancer cell lines to identify genes that are required for cell proliferation and/or viability. Cell line dependencies on 11,000 genes were interrogated by 5 shRNAs per gene. The proliferation effect of each shRNA in each cell line was assessed by transducing a population of 11M cells with one shRNA-virus per cell and determining the relative enrichment or depletion of each of the 54,000 shRNAs after 16 population doublings using Next Generation Sequencing. All the cell lines were screened using standardized conditions to best assess differential genetic dependencies across cell lines. When combined with genomic characterization of these cell lines, this dataset facilitates the linkage of genetic dependencies with specific cellular contexts (e.g., gene mutations or cell lineage). To enable such comparisons, we developed and provided a bioinformatics tool to identify linear and nonlinear correlations between these features.

We previously identified autoantibodies to the endocytic-associated protein Huntingtin-interacting protein 1-related (HIP1R) in diffuse large B-cell lymphoma (DLBCL) patients. HIP1R regulates internalization of cell surface receptors via endocytosis, a process relevant to many therapeutic strategies including CD20 targeting with rituximab. In this study, we characterized HIP1R expression patterns, investigated a mechanism of transcriptional regulation and its clinical relevance in DLBCL patients treated with immunochemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone, R-CHOP). HIP1R was preferentially expressed in germinal center B-cell-like DLBCL (P<0.0001) and inversely correlated with the activated B-cell-like DLBCL (ABC-DLBCL) associated transcription factor, Forkhead box P1 (FOXP1). HIP1R was confirmed as a direct FOXP1 target gene in ABC-DLBCL by FOXP1-targeted silencing and chromatin immunoprecipitation. Lower HIP1R protein expression (≤ 10% tumoral positivity) significantly correlated with inferior overall survival (OS, P=0.0003) and progression-free survival (PFS, P=0.0148) in R-CHOP-treated DLBCL patients (n=157). Reciprocal expression with ≥ 70% FOXP1 positivity defined FOXP1(hi)/HIP1R(lo) patients with particularly poor outcome (OS, P=0.0001; PFS, P=0.0016). In an independent R-CHOP-treated DLBCL (n=233) microarray data set, patients with transcript expression in lower quartile HIP1R and FOXP1(hi)/HIP1R(lo) subgroups exhibited worse OS, P=0.0044 and P=0.0004, respectively. HIP1R repression by FOXP1 is strongly associated with poor outcome, thus further understanding of FOXP1-HIP1R and/or endocytic signaling pathways might give rise to novel therapeutic options for DLBCL.

Fibroblast growth factor 23 (FGF23) is a hormone released primarily by osteocytes that regulates phosphate and vitamin D metabolism. Recent observational studies in humans suggest that circulating FGF23 is independently associated with cardiac hypertrophy and increased mortality, but it is unknown whether FGF23 can directly alter cardiac function. We found that FGF23 significantly increased cardiomyocyte cell size in vitro, the expression of gene markers of cardiac hypertrophy, and total protein content of cardiac muscle. In addition, FGFR1 and FGFR3 mRNA were the most abundantly expressed FGF receptors in cardiomyocytes, and the coreceptor α-klotho was expressed at very low levels. We tested an animal model of chronic kidney disease (Col4a3(-/-) mice) that has elevated serum FGF23. We found elevations in common hypertrophy gene markers in Col4a3(-/-) hearts compared with wild type but did not observe changes in wall thickness or cell size by week 10. However, the Col4a3(-/-) hearts did show reduced fractional shortening (-17%) and ejection fraction (-11%). Acute exposure of primary cardiomyocytes to FGF23 resulted in elevated intracellular Ca(2+) ([Ca(2+)](i); F/F(o) + 86%) which was blocked by verapamil pretreatment. FGF23 also increased ventricular muscle strip contractility (67%), which was inhibited by FGF receptor antagonism. We hypothesize that although FGF23 can acutely increase [Ca(2+)](i), chronically this may lead to decreases in contractile function or stimulate cardiac hypertrophy, as observed with other stress hormones. In conclusion, FGF23 is a novel bone/heart endocrine factor and may be an important mediator of cardiac Ca(2+) regulation and contractile function during chronic kidney disease.

Purpose: Approximately 5% of diffuse large B-cell lymphomas (DLBCLs) are double-hit lymphomas (DHLs) with translocations of both MYC and BCL2. DHLs are characterized by poor outcome. We tested whether DLBCLs with high expression of MYC protein and BCL2 protein share the clinical features and poor prognosis of DHLs., Patients and Methods: Paraffin-embedded lymphoma samples from 193 patients with de novo DLBCL who were uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) were studied using immunohistochemistry for MYC, BCL2, CD10, BCL6, and MUM1/interferon regulatory factor 4, and fluorescent in situ hybridization (FISH) for MYC and BCL2., Results: FISH analysis identified DHL in 6% of patients, who showed the expected poor overall survival (OS; P = .002). On the basis of immunohistochemical MYC and BCL2 expression, a double-hit score (DHS) was assigned to all patients with DLBCL. The DHS-2 group, defined by high expression of both MYC and BCL2 protein, comprised 29% of the patients. DHS 2 was significantly associated with lower complete response rate (P = .004), shorter OS (P < .001), and shorter progression-free survival (PFS; P < .001). The highly significant correlation with OS and PFS was maintained in multivariate models that controlled for the International Prognostic Index and the cell-of-origin subtype (OS, P < .001; PFS, P < .001). DHS was validated in an independent cohort of 116 patients who were treated with R-CHOP., Conclusion: The immunohistochemical DHS defined a large subset of DLBCLs with double-hit biology and was strongly associated with poor outcome in patients treated with R-CHOP.

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development-namely germinal center B-cell like and activated B-cell like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1 and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B cells. Cutoffs for each marker were obtained using receiver-operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1 and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy.

Determining the presence of MYC gene rearrangements is becoming an increasingly important part of the diagnostic workup in aggressive lymphoma. Cytogenetic MYC alterations aid in differentiating diffuse large B-cell lymphoma (DLBCL) from Burkitt lymphoma. In addition, MYC aberrations are associated with poor prognosis in DLBCL. Fluorescence in situ hybridization and karyotyping are standard tests for detecting MYC aberrations, but these techniques are laborious and expensive. Here, we studied MYC status of 219 DLBCLs and Burkitt lymphomas using fluorescence in situ hybridization, immunohistochemistry, and quantitative real-time polymerase chain reaction (QRT-PCR). Overall, 15% of the cases had an MYC break. QRT-PCR analysis of MYC expression showed that 72% of DLBCLs with an MYC break had aberrantly high or low levels of MYC transcript. Excluding the cases with aberrantly low MYC expression, we found a significant positive correlation between levels of MYC transcripts and MYC tumor cells; however, QRT-PCR is not readily applicable as a screening tool. Immunohistochemically, all tumors showed a nuclear staining pattern that was simple to evaluate. The percentage of MYC lymphoma cells correlated closely with MYC rearrangement status. In all, 93% of cases with an MYC break had ≥80% MYC cells, in contrast to 3% of nonrearranged cases (P<0.0001). Receiver operating characteristic curve analysis showed ≥70% MYC tumor cells to be the optimal cutoff (sensitivity=100%, specificity=93%). Area under the receiver operating characteristic curve was 0.992, indicating that immunostaining for Myc protein is an excellent screening test to predict whether an MYC rearrangement is present.

A prospective randomized study was conducted to determine if a design change in the articular surface geometry introduced in the Optetrak total knee to increase prosthetic joint conformity and further reduce polyethylene stress had any impact on implant survival, particularly when the all-polyethylene version of the implant was used. Forty-seven patients undergoing bilateral simultaneous total knee arthroplasties were randomized for the side, receiving an all-polyethylene tibial component and followed up for a mean 11.6 years. Survival rates for the all-polyethylene and metal-backed modular versions of the implant were both 98%, excluding a single case of deep infection. Survival rates with revision for aseptic loosening as an end point were 100%. The increase in tibial and femoral radii in the coronal plane of the Optetrak posterior stabilized knee did not result in a reduced implant survival rate in either the metal-backed modular or all-polyethylene versions of the implant., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Visual analytics is a new interdisciplinary field of study that calls for a more structured scientific approach to understanding the effects of interaction with complex graphical displays on human cognitive processes. Its primary goal is to support the design and evaluation of graphical information systems that better support cognitive processes in areas as diverse as scientific research and emergency management. The methodologies that make up this new field are as yet ill defined. This paper proposes a pathway for development of visual analytics as a translational cognitive science that bridges fundamental research in human/computer cognitive systems and design and evaluation of information systems in situ. Achieving this goal will require the development of enhanced field methods for conceptual decomposition of human/computer cognitive systems that maps onto laboratory studies, and improved methods for conducting laboratory investigations that might better map onto real-world cognitive processes in technology-rich environments., (Copyright © 2011 Cognitive Science Society, Inc.)

Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.

Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.

Introducing a research program into an echocardiography clinical practice can pose many challenges. Some initial factors to consider are the possible effects on the current clinical schedule and the equipment and personnel resources required to support the research projects. More importantly, how can an organization successfully complete reliable and accurate research projects? Here, we describe our experience with establishing an echocardiography research center within our clinical echocardiography practice. In addition, we identify key staff roles, highlight our current research practice methods, and suggest essential components that may prove advantageous when incorporating echocardiography research into a clinical practice.

Reported in this study are the experimental design and results of an immunosensor for the detection of the explosive, 2,4,6-trinitrotoluene (TNT) in seawater using a reversed-displacement format. This reversed-displacement immunosensor methodology has successfully measured TNT in seawater by direct injection, eliminating the need for preconcentration or pretreatment of samples. A microcolumn containing an Affi-Gel resin derivatized with a 2,4,6-trinitrobenzene (TNB) moiety and a fluorophore-labeled anti-TNT antibody composed the immunoassay reactive chamber. Fluorophore-labeled anti-TNT antibody was incubated with the modified Affi-Gel resin until binding equilibrium was reached. Under a constant flow, samples containing TNT were introduced into the flow stream displacing the fluorophore-labeled TNT antibody. Limits of detection were 2.5ng/mL or part-per-billion (ppb) for TNT in saline buffer and 25ppb in seawater with an analysis time of 10 min. Two anti-TNT antibodies with differing binding affinities were compared in the reversed-displacement assay format, and a correlation between affinity and detection limits was observed. Furthermore, we have demonstrated that the reversed-displacement format can be used to screen seawater samples containing TNT, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.

A capillary biosensor is demonstrated which uses the waveguiding properties of the capillary to integrate the signal over an increased surface area without simultaneously increasing the background noise from the detector. This biosensor achieves limits of detection of 30-50 pg/mL in immunoassays using a diode laser for excitation and a PMT for detection. This is approximately 2 orders of magnitude greater sensitivity than was achieved using the same immunoassay reagents in a fiber optic biosensor or a planar array biosensor. Two different approaches to using the capillaries as immunosensors are described, either of which could be adapted for multianalyte sensing.

Over a 5-year period at a single institution, 164 endoprostheses were used for the treatment of displaced femoral neck fractures. Soft-tissue restraint preservation and repair (hip capsule and labrum) is recommended to enhance stability after endoprosthesis placement. When dislocation does occur, however, this soft tissue may become interposed after closed reduction. This occurred in 2 of the dislocations reported here. In these 2 dislocations, despite the widened joint space, the range of motion was stable. Neither of these cases had a subsequent dislocation, and after 1 month, both had spontaneously narrowed their joint space back to the immediate postoperative state.

An uncemented titanium alloy stem with a corundum blast finish and an uncemented titanium fibermetal mesh socket were implanted in a series of 57 hips. These prostheses were selected for use in the youngest, most active, and/or heaviest candidates for total hip arthroplasty. Fifty hips were available for study at a minimum 60 months. At a mean 6 years, 92% of the hips were rated good or excellent. The mean Harris hip score was 92. One patient experienced mild thigh pain. The corundum blast finish was associated with reliable implant stability. Survival analysis predicted a 96% rate of implant survival at 92 months. Loss of bone density was rated mild, minimal, or none in 88% of the hips. Three hips developed severe bone loss due to systemic disease. Polyethylene wear was measurable in 86% of the hips. Twenty hips developed focal proximal femoral bone erosions. One hip had endosteal cavitation distal to zone 7. The presence of proximal femoral erosions or endosteal cavitation correlated positively with the presence of measurable polyethylene wear. The limited and proximal distribution of femoral bone erosion despite evidence of extensive polyethylene wear suggested that bone apposition to the corundum blast finish resulted in a barrier to migration of wear debris.

Fifty-one Cementless Spotorno (CLS, Protek A. G. Berne) stems were implanted in 43 patients with either a Harris Galante (Zimmer, Warsaw, IN) socket or bipolar head. Patients were evaluated at a mean of 31 months. Eighty percent of the hips were in patients who were less than 50 years of age or weighed more than 80 kg. The CLS stem achieved initial stability by wedging a proximally fluted, straight stem into a retained bed of femoral trabecular and cortical bone. Distal canal fill was avoided. The postoperative mean Harris hip score was 95. Eighty percent of the hips were rated excellent, 16% good, 2% fair, and 2% poor. No stem required revision. Six percent had slight, occasional thigh pain. No patient had mild, moderate, or severe thigh pain. Six percent had a limp related to the operated hip. Fifty-three percent of the hips developed a radiographic appearance of bone apposition at the stem tip. Fifty-five percent of the hips had some reduction in proximal bone density. These changes suggested that as bone remodeling occurred, the initial proximal load transfer situation expected from the CLS stem design changed to include some distal load transfer resulting in proximal stress shielding. Ninety-four percent of the hips had either no change in femoral bone density or only patchy loss of density isolated to zone 7. A high dislocation rate was attributed to an unfavorable head-to-neck diameter ratio, a valgus neck shaft angle, and a patient population capable of excellent hip motion.