Genome-wide open reading frame profiling identifies fibroblast growth factor signaling as a driver of PD-L1 expression in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCC) are associated with significant treatment-related morbidity and poor disease-free and disease-specific survival, especially in the recurrent and metastatic (R/M HNSCC) setting. Inhibition of the programmed death-1/ligand-1 (PD-1/PD-L1) immune checkpoint is accepted as a first-line treatment strategy for R/M HNSCC and has expanded into the neoadjuvant, definitive, and adjuvant settings. To understand cellular signals modulating the PD-L1 in HNSCC, we profiled a HNSCC cell-line with a genome-wide open reading frame (ORF) library of 17,000 individual constructs (14,000 unique genes). We identified 335 ORFs enriched in PD-L1high cells and independently validated five of these ORFs (FGF6, IL17A, CD300C, KLR1C and NFKBIA) as drivers of PD-L1 upregulation. We showed that exogenous FGF ligand is sufficient to induce PD-L1 expression in multiple HNSCC cell lines and human immature dendritic cells. Accordingly, overexpression of FGFR1, FGFR3 or the FGFR3 S249C and D786N mutants common to HNSCC tumors also induced PD-L1 overexpression on tumor cells. Small molecule inhibition of FGF signaling abrogated PD-L1 upregulation in these models and also blocked "classical" IFNγ-regulated PD-L1 expression in a STAT1-independent manner. Finally, we found that FGF specifically upregulated a glycosylated form of PD-L1 in our study, and exogenous FGF led to concomitant upregulation of glycosyltransferases that may stabilize PD-L1 on the surface of HNSCC cells. Taken together, our study supports a potential role for FGF/FGFR pathway signaling as a mechanism driving immune escape and rationalizes further exploration of novel combination therapies to improve clinical responses to PD-1/PD-L1 axis inhibition in HNSCC.

SearcHPV: A novel approach to identify and assemble human papillomavirus-host genomic integration events in cancer.

BACKGROUND: Human papillomavirus (HPV) is a well-established driver of malignant transformation at a number of sites, including head and neck, cervical, vulvar, anorectal, and penile squamous cell carcinomas; however, the impact of HPV integration into the host human genome on this process remains largely unresolved. This is due to the technical challenge of identifying HPV integration sites, which includes limitations of existing informatics approaches to discovering viral-host breakpoints from low-read-coverage sequencing data. METHODS: To overcome this limitation, the authors developed SearcHPV, a new HPV detection pipeline based on targeted capture technology, and applied the algorithm to targeted capture data. They performed an integrated analysis of SearcHPV-defined breakpoints with genome-wide linked-read sequencing to identify potential HPV-related structural variations. RESULTS: Through an analysis of HPV+ models, the authors showed that SearcHPV detected HPV-host integration sites with a higher sensitivity and specificity than 2 other commonly used HPV detection callers. SearcHPV uncovered HPV integration sites adjacent to known cancer-related genes, including TP63, MYC, and TRAF2, and near regions of large structural variation. The authors further validated the junction contig assembly feature of SearcHPV, which helped to accurately identify viral-host junction breakpoint sequences. They found that viral integration occurred through a variety of DNA repair mechanisms, including nonhomologous end joining, alternative end joining, and microhomology-mediated repair. CONCLUSIONS: In summary, SearcHPV is a new optimized tool for the accurate detection of HPV-human integration sites from targeted capture DNA sequencing data.

Association of human papillomavirus integration with better patient outcomes in oropharyngeal squamous cell carcinoma.

BACKGROUND: The molecular drivers of human papillomavirus-related head and neck squamous cell carcinoma (HPV + HNSCC) are not entirely understood. This study evaluated the relationship between HPV integration, expression of E6/E7, and patient outcomes in p16+ HNSCCs. METHODS: HPV type was determined by HPV PCR-MassArray, and integration was called using detection of integrated papillomavirus sequences polymerase chain reaction (PCR). We investigated whether fusion transcripts were produced by reverse transcriptase polymerase chain reaction (RT-PCR). E6/E7 expression was assessed by quantitative RT-PCR. We assessed if there was a relationship between integration and E6/E7 expression, clinical variables, or patient outcomes. RESULTS: Most samples demonstrated HPV integration, which sometimes resulted in a fusion transcript. HPV integration was positively correlated with age at diagnosis and E6/E7 expression. There was a significant difference in survival between patients with vs without integration. CONCLUSIONS: Contrary to previous reports, HPV integration was associated with improved patient survival. Therefore, HPV integration may act as a molecular marker of good prognosis.

Efficacy of axitinib in metastatic head and neck cancer with novel radiographic response criteria.

BACKGROUND: There are limited treatment options for unresectable recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Vascular endothelial growth factor is of significant interest for targeted therapy in R/M HNSCC because of its central role in tumorigenesis and immunosuppression. Axitinib is a potent inhibitor of vascular endothelial growth factor receptor (VEGFR) 1 , VEGFR2, VEGFR3, platelet-derived growth factor receptor, as well as c-kit and offers such an approach. METHODS: This article reports the results of a phase 2 trial evaluating axitinib in R/M HNSCC according to the Choi criteria for radiographic response assessment. The primary endpoint of this trial was 6-month overall survival. RESULTS: Twenty-nine patients were enrolled, and 28 were evaluable for a response. Patients were heavily pretreated with 61% having had at least 1 previous systemic treatment in the metastatic setting (range, 0-5). The median overall survival of 9.8 months and the 6-month overall survival rate of 70% met the protocol-defined criteria for clinical efficacy. The best overall response rate was 42%. Correlative analyses demonstrated that PI3K signaling pathway alterations were associated with an increased response to therapy (75% vs 17%). A marked response to therapy was seen in a subgroup of patients who were treated with an immune checkpoint inhibitor after progression on axitinib. CONCLUSIONS: Treatment with axitinib is associated with improved survival in patients with heavily pretreated head and neck cancer, and PI3K pathway alterations may serve as a biomarker for response. Further investigation is warranted to evaluate axitinib in biomarker-selected populations, especially in combination with immune checkpoint inhibitor therapy. LAY SUMMARY: Metastatic head and neck squamous cancer is an incurable disease with limited treatment options and a poor prognosis. This study is the first to demonstrate that the targeted oral drug axitinib improves survival in patients with heavily pretreated metastatic head and neck cancer. Furthermore, patients whose tumors have specific mutations derive the greatest benefit from therapy. The investigation of axitinib alone or in combination with immunotherapy in a genomic biomarker-selected population is warranted.

A survey of stress, burnout and well-being in UK dentists.

Introduction: It is well established that dentistry is a stressful profession, primarily due to the nature and working conditions in the dental surgery. With dramatic changes taking place in the profession in recent years it is important to establish the impact this has on dentists' well-being. Aims: To determine the levels of stress and burnout in UK dentists and how this relates to well-being and identify the sources of work-related stress dentists report in different fields of practice. Materials and method: An online survey comprising of validated measures examining stress, burnout and well-being in dentists was administered to British Dental Association (BDA) members and non-members. Results: Valid responses were received from 2053 respondents. Dentists working in the UK exhibit high levels of stress and burnout and low well-being. General dental practitioners (GDPs) seem to be particularly affected. Issues relating to regulation and fear of litigation were deemed to be the most stressful aspects of being a dentist. Conclusions: The findings from this study build upon existing research showing that dentistry is a stressful profession. The sources of this stress appear to have shifted over the years, highlighting the changing landscape of dentistry. Interventions should focus on addressing these stressors by making changes to the working conditions of dentists.

Associates and their working environment: a comparison of corporate and non-corporate associates.

Introduction The share of the dental market held by corporate bodies continues to increase. With the profession currently facing many challenges it is important to understand their place in the profession and their effect.Aim This exploratory study aims to provide an insight into dental associates in relation to their working environment. Specifically, the differences between working in corporate and non-corporate environments in England.Materials and methods Secondary analysis of a self-report questionnaire examining demographics, pay, working conditions, job satisfaction and morale using a combination of closed and open-ended responses sent to randomly selected associate dentists who are BDA members. Responses from associates working in England solely in either the corporate or non-corporate sector were analysed.Results Significant differences were seen between associates working in corporate practice when compared to those in non-corporates practice, for example, significantly less corporate associates were female and corporate associates reported relatively lower levels of autonomy and control.Discussion The differences seen between sectors could be related to rationalisation and should this be the reality it could have far reaching effects on the profession and its ability to manage itself.Conclusion This study highlights some differences between the corporate and non-corporate dental sectors. Further work is needed to build a deeper understanding of the sector.

New insights regarding rationale, therapeutic target and dose of hemofiltration and hybrid therapies in septic acute kidney injury.

Mediator removal from tissue (capillary blood compartment, CABC) and transport to the central circulation (central blood compartment, CEBC) must be effective. Effectiveness through a passive mechanism seems unlikely as the surface of CEBC (30 m(2)) is smaller than CABC (300 m(2)) whereby the former will be a limiting factor in passive transport. According to studies, a high exchange volume can induce an 80-fold increase in lymphatic flow. This results in displacement (active transport) of mediators to CEBC. Recent studies have shown that the delivered dose constitutes the mainstay of continuous renal replacement therapy. However, these results are not likely to change the recommendation: 35 ml/kg/h, adjusted for predilution, in septic acute kidney injury (AKI). Recently, studies were focusing on global intensive care unit AKI. In non-septic AKI, those studies show that 20-25 ml/kg/h was optimal. The DO-RE-MI trial underscored the importance of delivery which could be obtained by targeting doses between 5 and 10 ml/kg/h higher than prescribed. Until the IVOIRE trial becomes available, septic AKI should be treated by continuous veno-venous hemofiltration at 35 ml/kg/h. In non-septic AKI, 25 ml/kg/h remains optimal.

Septic acute kidney injury: the culprit is inflammatory apoptosis rather than ischemic necrosis.

For a long time, acute kidney injury (AKI) was considered to be a primarily hemodynamic condition characterized by a reduction of renal blood flow, induced by either cardiogenic or distributive (septic) shock. Consequently, all efforts to treat AKI were essentially concentrated on increasing renal flow by enhancing cardiac flow output and perfusion pressure. At the beginning of this decade, Bellomo and co-workers produced new and intriguing data in an animal model of septic AKI that undermined existing concepts. They observed that medullar and cortical renal blood flow were both maintained and even increased in septic shock, underscoring that septic AKI was a totally different physiological phenomenon than nonseptic AKI. Also, apoptosis was found to play a more important role in sepsis and septic shock than pure necrosis. Despite these findings, the role of apoptosis as a main mechanism of organ dysfunction remains topic of debate.

Continuous hemofiltration in 2009: what is new for clinicians regarding pathophysiology, preferred technique and recommended dose?

In the last years, publications have questioned the classical dose of 35 ml/kg, but are those studies strong enough in terms of scientific power in order to change our practice? We will try to settle some recommendations for clinicians. Manipulation of dose, porosity, and combinations have yielded promising findings. However, conclusive evidence based on randomized trials remains scarce, limiting the practical implementation in daily practice. From the few designed studies, it is safe to say that optimization of delivered dose has a proven positive effect. An ultrafiltration rate of around 35 ml/kg/h, with adjustment for predilution, can be recommended for the septic patient. Recent studies do not have enough power to change this recommendation in view of its shortcomings. Finally the recommendation is to keep going with a continuous technique, a pure continuous veno-venous hemofiltration mode, and at a dose of 35 ml/kg/h while waiting for other studies to be published.

Aptitude, achievement and competence in medicine: a latent variable path model.

To develop and test a latent variable path model of general achievement, aptitude for medicine and competence in medicine employing data from the Medical College Admission Test (MCAT), pre-medical undergraduate grade point average (UGPA) and demographic characteristics for competence in pre-clinical and measures of competence (United States Licensure Examination {USMLE} Steps 1, 2, and 3). Data were gathered on 839,710 participants from 1991 to 2000 on demographic and school variables, UGPA, MCAT subtest scores and Steps 1, 2, and 3 of the United Stated Licensure Examination (USMLE). However, subsets of the total 839,710 participants included in the database were used for various analyses and the testing of a latent variable path model (LVPA). A number of preliminary descriptive and inferential techniques were used to confirm previous hypotheses and stated relationships amongst the variables of interest to the present study. Through development and testing of a latent variable path model, three latent variables measured by UGPA (general achievement), subscales of the MCAT (aptitude for medicine), and Steps 1, 2, and 3 of the USMLE (competence in medicine) were identified which resulted in a comparative fit index = .932 of the model to a large sample (n = 20,714). In a confirmatory latent variable path model we were able to identify theoretical constructs, aptitude for medicine, general achievement, and competence in medicine and their interrelationships. These are distinct but interrelated latent variables.

Evaluation of an automated repetitive sequence-based PCR system for subtyping Enterobacter sakazakii.

Enterobacter sakazakii is regarded as a ubiquitous organism that can be isolated from a wide range of foods and environments. Infection in at-risk infants has been epidemiologically linked to the consumption of contaminated powdered infant formula. Preventing the dissemination of this pathogen in a powdered infant formula manufacturing facility is an important step in ensuring consumer confidence in a given brand together with the protection of the health status of a vulnerable population. In this study we report the application of a repetitive sequence-based PCR typing method to subtype a previously well-characterized collection of E. sakazakii isolates of diverse origin. While both methods successfully discriminated between the collection of isolates, repetitive sequence-based PCR identified 65 types, whereas pulsed-field gel electrophoresis identified 110 types showing > or =95% similarity. The method was quick and easy to perform, and our data demonstrated the utility and value of this approach to monitor in-process contamination, which could potentially contribute to a reduction in the transmission of E. sakazakii.

[High-volume hemofiltrations].

Almost 10 years ago, standard hemofiltration (HF) was carried out at an ultrafiltration (UF) rate of not more than 2 1/hour and only a predilution variant was usually used. However, after Ronco published the results of his study early in the 20th century the situation changed as this study provided evidence that increasing an UF rate up to 35 ml/kg/hr had a positive effect in patients with acute renal failure (ARF). At the same time, there was a new standard for the determination of high-volume HF (HVHF) and new concepts generalized in this paper have emerged. Moreover, two different procedures of HVHF: (1) a continuous high-volume technique providing a rate of 50-70 ml/kg/hr during 24 hours; (2) an intermittent high-volume technique, the so-called HVHF with short courses of treatment, a very high volume at a rate of 100-120 ml/kg/hr during 4-8 hours. Later on this technique came to be called pulse HVHF. Both methods are designated by the abbreviation HVHF, but their concepts and results are slightly different, as will be discussed below. As for the higher doses of plasma replacement therapy, it is necessary to put the results of two recently published studies into routine clinical practice of continuous vein-venous HF (CVVHF) as soon as possible just in 2008. The performed studies suggest the most important role of adequate dosage of CVVHF and show that the use of a dose of 35 ml/kg/hr in critically ill patients with ARF increased survival by almost 20%. These two studies contain today's most vivid evidence, but one awaits the results of other studies that confirm (or do not confirm) these conclusions. In the world where more importance is attached to evidence-based medicine, two first-level studies will yield Stage A recommendations that may be guided by any intensive care specialist using CVVHF in expectation of the results of on-going investigations. Nevertheless, many difficulties are to be surmounted while introducing new procedures into routine intensive care practice. The most cardinal problems are those associated with requirements for blood flow via vascular access with pre- and postdilution procedures, with the type of employed membranes and substituting liquids, and with a possible need for concomitant dialysis. From the strictly practical standpoint, introduction of these changes into routine intensive care practice may be extremely difficult. This will undoubtedly require that physicians and nurses should make joint effects.

Assessment of non-cognitive traits through the admissions multiple mini-interview.

CONTEXT: Contemporary studies have shown that traditional medical school admissions interviews have strong face validity but provide evidence for only low reliability and validity. As a result, they do not provide a standardised, defensible and fair process for all applicants. METHODS: In 2006, applicants to the University of Calgary Medical School were interviewed using the multiple mini-interview (MMI). This interview process consisted of 9, 8-minute stations where applicants were presented with scenarios they were then asked to discuss. This was followed by a single 8-minute station that allowed the applicant to discuss why he or she should be admitted to our medical school. Sociodemographic and station assessment data provided for each applicant were analysed to determine whether the MMI was a valid and reliable assessment of the non-cognitive attributes, distinguished between the non-cognitive attributes, and discriminated between those accepted and those placed on the waitlist (waiting list). We also assessed whether applicant sociodemographic characteristics were associated with acceptance or waitlist status. RESULTS: Cronbach's alpha for each station ranged from 0.97-0.98. Low correlations between stations and the factor analysis suggest each station assessed different attributes. There were significant differences in scores between those accepted and those on the waitlist. Sociodemographic differences were not associated with status on acceptance or waiting lists. DISCUSSION: The MMI is able to assess different non-cognitive attributes and our study provides additional evidence for its reliability and validity. The MMI offers a fairer and more defensible assessment of applicants to medical school than the traditional interview.

Characterization of thioredoxin y, a new type of thioredoxin identified in the genome of Chlamydomonas reinhardtii.

The sequencing of the Arabidopsis genome revealed a multiplicity of thioredoxins (TRX), ubiquitous protein disulfide oxido-reductases. We have analyzed the TRX family in the genome of the unicellular green alga Chlamydomonas reinhardtii and identified eight different thioredoxins for which we have cloned and sequenced the corresponding cDNAs. One of these TRXs represents a new type that we named TRX y. This most probably chloroplastic TRX is highly conserved in photosynthetic organisms. The biochemical characterization of the recombinant protein shows that it exhibits a thermal stability profile and specificity toward target enzymes completely different from those of TRXs characterized so far.

Murine dendritic cells derived from myeloid progenitors of the thymus are unable to produce bioactive IL-12p70.

Dendritic cells (DC) are present at low density in the thymus where they mediate negative selection of self-reactive thymocytes. Previous reports suggest that thymic DC (TDC) are a single population of lymphoid-related DC. In this study, we documented the presence in the adult mouse thymus of an additional population of TDC exhibiting a myeloid phenotype (CD11c(+) CD8alpha(-) CD11b(+)). This population, which can be purified, represented approximately 20% of the total TDC and differs from the population of lymphoid TDC (CD11c(+) CD8(+) CD11b(-)) by its incapacity to produce IL-12p70 under double stimulation by LPS and anti-CD40. Furthermore, using an original culture system allowing expansion of DC from myeloid progenitors, we demonstrated that DC exhibiting a similar myeloid phenotype can be derived from a common DC/macrophage progenitor resident in the adult mouse thymus. We found that, in contrast with myeloid splenic DC expanded in the same conditions, these cultured TDC were unable to produce IL-12p70 under double stimulation by LPS and anti-CD40 or LPS and IFN-gamma. Thus, our results suggest that 1) adult mouse thymus contains at least two phenotypically and functionally distinct populations of DC; and 2) cultured myeloid DC derived from thymus and spleen differ by their ability to produce IL-12p70. The mechanisms underlying the differences in IL-12-secreting capacities of the cultured splenic and thymic DC are under current investigation.

Evaluation of acute normovolemic hemodilution for surgical repair of craniosynostosis.

This clinical report investigated the potential benefit of acute normovolemic hemodilution (ANH) as a blood-saving technique in the surgical repair of craniosynostosis. Over a 4-year period, 34 healthy children undergoing surgical repair of scaphocephaly or pachycephaly were randomly assigned to two groups of 17 patients each. Patients of the first group (ANH group) were submitted to ANH (target Ht: 25%) immediately before surgery and patients of the second group (Control group) were not. During surgery, estimated blood loss was compensated with a 5% albumin solution and no autologous or homologous blood was transfused. At the end of surgery, intraoperative blood loss (mean +/- SD) calculated on the basis of the Ht value and the children weight was 21.3+/-8% of the estimated blood volume (EBV) in the ANH group and 24+/-6.6% in the Control group. Children of the ANH group received their autologous blood (18.9+/-3.3% of EBV) systematically at the end of surgery. In the postoperative period, homologous blood was transfused when the Ht value was equal or less than 21%. Both groups were comparable regarding age, weight, type of craniosynostosis, duration of surgery, EBV, and preoperative Ht value. No difference was observed between ANH and Control groups in the number of patients who received homologous blood (15/17 and 14/17, respectively), in the amount of homologous blood transfused (17+/-4.7% and 19.6+/-6.3% of the EBV, respectively), and in the Ht value before hospital discharge (29.4+/-5.0% and 30.7+/-4.9%, respectively). In conclusion, this report suggests that ANH reduces neither the incidence of homologous transfusion nor the amount of homologous blood transfused in this series of children undergoing surgical repair of craniosynostosis.

Mimicking lipid-binding-induced conformational changes in the human apolipoprotein E N-terminal receptor binding domain effects of low pH and propanol.

We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1-191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1-191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross-linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu-C, as is the case for apo E3(1-191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n-propanol increases the amount of calculated alpha-helical structure (42-65%) and alters the tertiary structure of the protein although not as much as when apo E3(1-191) is incorporated into lipid bicelles. In the absence of n-propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n-propanol at pH 4.5 increases the Stokes radius of apo E3(1-191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0-4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu-C. As assessed by far and near UV circular dichroism, the addition of 30% n-propanol at pH 4.5, in contrast to pH 7, markedly increases the alpha-helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1-191) into lipid bicelles. The results suggest that a combination of n-propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids.

Referral and screening patterns at a clinic for anxiety disorders; implications for the planning of clinical studies.

During a two year period (1988-1990), 302 patients who were referred to our anxiety disorder clinic were evaluated and diagnosed according to a screening procedure which will be described. Of these initial referrals, 171 met a DSM-3-R diagnosis of panic disorder (with or without agorophobia) or generalized anxiety disorder; 81 patients (27% of the initial referrals) participated in a double blind placebo controlled clinical trial with a dropout rate of 12%. The implications for the planning and design of referral and screening strategies at research oriented specialized clinics are discussed.