Gestational choline supplementation ameliorates T-maze reversal learning deficits induced by first trimester binge alcohol exposure in weanling lambs.

In rodent models of fetal alcohol spectrum disorders (FASD), cognitive deficits are implicated in impaired T-maze spatial reversal learning. Rat studies have indicated supplemental administration of choline during the developmental period of alcohol exposure can ameliorate spatial reversal deficits. This study tested whether beneficial effects of prenatal choline supplementation could be confirmed in a sheep model of binge exposure in the first trimester equivalent. Two hypotheses were tested: 1) alcohol exposure would produce deficits in reversal of a T-maze position discrimination; and 2) gestational dietary supplementation of choline would ameliorate those deficits. Mated ewes were assigned to one of seven groups-a normal control (NC) group or one of six infusion treatment groups: saline control (SC; isotonic saline), saline control plus choline (SC-CH; isotonic saline plus choline, 10 mg/kg administered orally throughout each day of gestation), binge alcohol (BA; 1.75 g/kg alcohol per infusion day), binge alcohol plus choline (BA-CH; 1.75 g/kg/day alcohol plus choline), heavy binge alcohol (HBA; 2.5 g/kg/day alcohol), or heavy binge alcohol plus choline (HBA-CH; 2.5 g/kg/day alcohol plus choline). The alcohol infusions modeled a weekend binge drinking pattern over the first trimester-equivalent (gestational day 4-41). T-maze training began at 12 weeks of age, with daily sessions occurring 5 days/week. Lambs were given five days of habituation training, followed by five days of position discrimination training (3 trials per daily session, intertrial interval of 3 hours, reinforced side randomly assigned across subjects). Lambs were then given 10 days of training on the reversal task. There was no difference among groups during acquisition. Alcohol impaired reversal learning, and choline supplementation mitigated these deficits in the HBA-CH group. These results suggest that maternal dietary choline supplementation can ameliorate or prevent some impairments of executive function in a sheep model of FASD.

Design and development of microformulations for rapid release of small molecules and oligonucleotides.

A systemic delivery of therapeutics frequently results in sub-optimal exposure of the targeted locus and undesired side effects. To address these challenges, a platform for local delivery of diverse therapeutics by remotely controlled magnetic micro-robots was introduced. The approach involves micro-formulation of active molecules using hydrogels that exhibit wide range of loading capabilities and predictable release kinetics. This work introduces two specific hydrogels based on thiol-maleimide and PEG-PLA-diacrylate chemistries that afford high, reliable and reproducible loading and release of several model molecules including doxorubicin, 25-mer poly-dT oligonucleotide and a 5.4 kBp GFP DNA plasmid. The described formulations are suitable for micro-dosing using both conventional or remote delivery devices.

Laparoscopy uptake for paediatric appendicectomy: a comparison of general surgeons versus specialist paediatric surgeons in England from 1997 to 2015.

INTRODUCTION: Laparoscopy is used in as many as 95% of adult appendicectomies. There is level I evidence showing that it reduces wound infection, postoperative ileus and length of inpatient stay in children compared with the open approach. The aim of this study was to report the uptake of laparoscopy for paediatric appendicectomy in England and to determine whether this was similar for general surgeons (GS) and specialist paediatric surgeons (SPS). METHODS: Hospital Episode Statistics data were obtained for all children aged <16 years who had an OPCS 4.6 code for emergency appendicectomy from 1997 to 2015 (18 years). Data are analysed to compare rate of laparoscopic vs open procedures for GS and SPS over time and to investigate factors associated with the use of laparoscopy. RESULTS: There were 196,987 appendicectomies and where specialty was available, 133,709 (79%) cases were undertaken by GS and 35,141 (21%) by SPS. The rate of cases undertaken with laparoscopy for both specialties combined increased from 0.8% in 1998 to 50% in 2014 (p<0.0001). In 2014, this rate was 41% for GS compared with 71% for SPS (p<0.0001). Female gender (odds ratio (OR)=1.84, 95% confidence interval (CI) 1.80-1.90), increasing age (OR=1.18, 95% CI 1.18-1.19 per year) and treatment by SPS (OR=3.71, 95% CI 3.60-3.82) were all factors positively associated with use of laparoscopy in multivariate analysis. CONCLUSIONS: There has been a vast increase in the proportion of appendicectomies undertaken laparoscopically in children. Despite adjusting for patient factors, laparoscopy was used significantly less by GS when compared with SPS. This difference is most apparent in younger children.

Piloting an integrated approach for estimation of environmental risk of Schistosoma haematobium infections in pre-school-aged children and their mothers at Barombi Kotto, Cameroon.

Within schistosomiasis control, assessing environmental risk of currently non-treated demographic groups e.g. pre-school-aged children (PSAC) and their mothers is important. We conducted a pilot micro-epidemiological assessment at the crater lake of Barombi Kotto, Cameroon with GPS tracking and infection data from 12 PSAC-mother pairs (n = 24) overlaid against environmental sampling inclusive of snail, parasite and water-use information. Several high-risk locations or 'hotspots' with elevated water contact, increased intermediate snail host densities and detectable schistosome environmental DNA (eDNA) were identified. Exposure between PSAC and mother pairs was temporally and spatially associated, suggesting interventions which can benefit both groups simultaneously might be feasible. When attempting to interrupt parasite transmission in future, overlaid maps of snail, parasite and water contact data can guide fine-scale spatial targeting of environmental interventions.

Repair of oesophageal atresia by consultants and supervised trainees results in similar outcomes.

INTRODUCTION: Consultants and trainees require exposure to complex cases for maintaining and gaining operative experience. Oesophageal atresia (OA) repair is a neonatal surgical procedure with indicative numbers for completion of training. A conflict of interest may exist between adequate training, maintaining consultant experience and achieving good outcomes. We aimed to review outcomes of procedures performed primarily by trainees and those performed by consultants. METHODS: We carried out a retrospective case note review of all consecutive infants who underwent surgical repair of OA with distal tracheooesophageal fistula (TOF) between January 1994 and December 2014 at our institution. Only cases that underwent primary oesophageal anastomosis were included. Surgical outcomes were compared between cases that had a trainee and those that had a consultant listed as the primary operator. RESULTS: One hundred and twenty-two cases were included. A total of 52 procedures were performed by trainees, and 68 by consultants. Two cases were undeterminable and excluded. Infant demographics, clinical characteristics and duration of follow-up were similar between groups. All infants survived to discharge. Procedures performed by trainees and those performed by consultants as primary operators had a similar incidence of postoperative pneumothorax (trainees 4, consultants 3; p=0.46), anastomotic leak (trainees 5, consultants 3; p=0.29) and recurrent TOF (trainees 0, consultants 2; p=0.5). Overall 52% of cases had an anastomotic dilatation during follow-up, with no difference between the trainee and consultant groups (50% vs 53%; p=0.85). CONCLUSIONS: Surgical outcomes for repair of OA/TOF are not adversely affected by trainee operating. Trainees with appropriate skills should perform supervised OA/TOF repair. These data are important for understanding the interrelationship between provision of training and surgical outcomes.

Role of the virtual reality simulator (ScanTrainer) as a multidisciplinary training tool in transvaginal ultrasound: A systematic review and narrative synthesis.

OBJECTIVES: This systematic review investigates the role of the ScanTrainer as a virtual reality training simulator and its impact on the scanning skills in transvaginal ultrasound of novice ultrasound practitioners. KEY FINDINGS: After searching ten databases for studies incorporating the simulator as a part of the learning/training process, ten out of 684 studies met the eligibility criteria and were included in the review. The analysis of the textual findings using narrative synthesis approach resulted in four themes: Validation (assessment of the validity of the simulator), Learning (using the simulator as a learning tool), Perspectives (the perceptions of participants trained on the simulator), and Transferable skills (skills developed on the simulator can transfer to clinical practice). CONCLUSION: Although literature indicates that the simulator is valuable as a training/learning tool, there is insufficient evidence of measurable effects on clinical practice of simulator usage by different healthcare professions.

Maxillary sinus augmentation using chairside bone marrow aspirate concentrates for implant site development: a systematic review of histomorphometric studies.

Maxillary sinus pneumatization following dental tooth extractions and maxillary alveolar bone resorption frequently leaves inadequate bone levels for implant placement. The objectives of this systematic review are to evaluate the effects of bone marrow aspirate concentrates (BMACs) used in maxillary sinus augmentation for implant site development.A systematic search was conducted using PubMed, EMBASE, Web of Science, Cochrane Library, and Google Scholar for studies which histomorphometrically evaluated the efficacy of BMACs and BMAC-enriched biomaterials in sinus floor elevation. Six studies were selected, and the risk of bias was evaluated.Reported ranges of vital mineralized tissue for the BMAC groups for the selected studies were 34.63-55.15% compared to 27.30% for control groups. For vital mineralized bone, these studies reported variable statistical significance. At 3-4 months, new bone formation for BMAC groups with controls using no BMAC was 7.4-12.6% and for the control groups was 9.45-14.3%. At 6 months, new bone formation for BMAC groups was 13.5-14.12% and for control groups was 10.41-13.9%. For new bone formation, these studies reported no significant difference between test and control and between 3 and 6 months histologic evaluation.Within the limits of this systematic review, the chairside method to harvest BMAC produced similar implant survival and new bone formation compared to the laboratory FICOLL group, without the additional cost and time of laboratory cell isolation techniques. The iliac crest or tibia origins, single or double centrifugation, for BMAC do not appear to be a factor for implant survival or bone formation. Although some favorable outcomes were reported, the increase in new bone formation using chairside-harvested BMAC compared to control is not predictably more significant across studies.Clinically, new bone formation in the maxillary sinus is not always contingent on the presence of BMAC. The novelty of this method requires more future studies.

Age and experience dependent changes in Egr-1 expression during the ontogeny of the context preexposure facilitation effect (CPFE).

The context preexposure facilitation effect (CPFE) is a variant of contextual fear conditioning in which acquisition of the contextual representation and association of the retrieved contextual memory with an immediate foot-shock are separated by 24 h. During the CPFE, learning- related expression patterns of the early growth response-1 gene (Egr-1) vary based on training phase and brain sub-region in adult and adolescent rats (Asok, Schreiber, Jablonski, Rosen, & Stanton, 2013; Schreiber, Asok, Jablonski, Rosen, & Stanton, 2014; Chakraborty, Asok, Stanton, & Rosen, 2016). The current experiments extended our previous findings by examining Egr-1 expression in infant (PD17) and juvenile (PD24) rats during the CPFE using preexposure protocols involving single-exposure (SE) or multiple-exposure (ME) to context. Following a 5 min preexposure to the training context (i.e. the SE protocol), Egr-1 expression in the medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC) and lateral nucleus of the amygdala (LA) was differentially increased in PD24 rats relative to PD17 rats. In contrast, increased Egr-1 expression following an immediate foot-shock (2s, 1.5 mA) did not differ between PD17 and PD24 rats, and was not learning-related. Interestingly, increasing the number of exposures to the training chamber on the preexposure day (i.e. ME protocol) altered training-day expression such that a learning-related increase in expression was observed in the mPFC in PD24 but not PD17 rats. Together, these results illustrate a clear maturation of Egr-1 expression that is both age- and experience-dependent. In addition, the data suggest that regional activity and plasticity within the mPFC on the preexposure but not the training day may contribute to the ontogenetic profile of the effect. Further studies are necessary to elucidate the causal role of sub-region-specific neuroplasticity in the ontogeny of the CPFE.

Maxillary Sinus Augmentation for Dental Implant Rehabilitation of the Edentulous Ridge: A Comprehensive Overview of Systematic Reviews.

OBJECTIVES: The objective of this systemic review was to perform a comprehensive overview of systematic reviews and meta-analyses of the maxillary sinus augmentation procedure for implant rehabilitation in humans. The following were evaluated in this overview: (1) anatomic variables affecting sinus augmentation, (2) histomorphometric analysis of the grafted sinus, (3) volumetric changes after sinus grafting, and (4) implant survival beyond 1 year. MATERIAL AND METHODS: Electronic databases were searched for systematic reviews and meta-analyses of implant-related sinus augmentation published from 1976 to September 2015. The studies selected must identify itself as a systemic review or meta-analysis in the title or abstract and must pertain to sinus augmentation. RESULTS: Thirty-three publications fulfilled the review criteria. The AMSTAR ratings for the 33 chosen reviews scored greater than 3 of 11, with 8 reviews scoring greater or equal to 8 of 11. CONCLUSION: The outcome of this overview suggested that the following will increase the success of sinus augmentation and survival of implants placed in the grafted sinus: (1) the use of barrier membranes over the lateral window when using a lateral approach to graft the sinus, (2) the use of particulate autogenous bone with or without other substitute graft materials, (3) sinus augmentation without the use of grafting materials may be considered provided that the space between the sinus membrane and floor can be maintained, (4) the use of rough-surfaced implants, (5) simultaneous implant placement with residual bone height greater than 4 mm, and (6) the cessation of smoking.

Antagonism of muscarinic acetylcholine receptors in medial prefrontal cortex disrupts the context preexposure facilitation effect.

Cholinergic function plays a role in a variant of context fear conditioning known as the context preexposure facilitation effect (CPFE; Robinson-Drummer, Dokovna, Heroux, & Stanton, 2016). In the CPFE, acquisition of a context representation, the context-shock association, and expression of context fear occur across successive phases, usually 24h apart. Systemic administration of scopolamine, a muscarinic acetylcholine receptor antagonist, prior to each phase (context preexposure, immediate-shock training, and testing) disrupts the CPFE in juvenile rats (Robinson-Drummer et al., 2016). Dorsal hippocampal (dHPC) cholinergic function contributes significantly to this effect, as local infusion of scopolamine into the dHPC prior to any individual phase of the CPFE produces a disruption identical to systemic administration (Robinson-Drummer et al., 2016). The current experiment extended these findings to another forebrain region implicated in the CPFE, the medial prefrontal cortex (mPFC). Adolescent rats received bilateral infusions of scopolamine (35µg/side) or PBS 10min before all three phases of the CPFE or only prior to a single phase. Intra-mPFC administration of scopolamine prior to all three phases significantly impaired fear conditioning suggesting that mPFC cholinergic function is necessary for successful CPFE performance. Analyses of the individual infusion days revealed a significant impairment of the CPFE when infusions occurred prior to preexposure or training (i.e. immediate footshock) but not prior to testing. In total, these findings suggests a role of mPFC cholinergic function in the acquisition and/or consolidation of a contextual representation and the context-shock association but not in retrieval or expression of fear memory. Implications for mPFC involvement in contextual fear conditioning and neurological dysfunction following neonatal alcohol exposure are discussed.

The Role of Spatial Statistics in the Control and Elimination of Neglected Tropical Diseases in Sub-Saharan Africa: A Focus on Human African Trypanosomiasis, Schistosomiasis and Lymphatic Filariasis.

Disease control and elimination programmes can benefit greatly from accurate information on the spatial variability of disease risk, particularly when risk is highly spatially heterogeneous. Due to advances in statistical methodology, coupled with the increased availability of geospatial technology, this information is becoming increasingly accessible. In this chapter we describe recent advancements in spatial methods associated with the analysis of disease data measured at the point-level and demonstrate their application to the control and elimination of neglected tropical diseases (NTDs). We further provide information on spatially referenced data sources and software that can be used to create NTD risk maps, concentrating on those that can be freely obtained. Examples relating to three NTDs affecting populations in sub-Saharan Africa are presented throughout the chapter, i.e., human African trypanosomiasis, schistosomiasis and lymphatic filariasis. These three diseases, with differing routes of transmission, control methods and level of spatial heterogeneity, demonstrate the flexibility and applicability of the methods described.

Duhamel versus transanal endorectal pull through (TERPT) for the surgical treatment of Hirschsprung's disease.

For the surgical treatment of Hirschsprung's disease, several surgical techniques are used to resect the distal aganglionic colon. Two frequently used techniques are the Duhamel procedure and the transanal endorectal pull-through procedure. During the '8th Pediatric Colorectal Course' in Nijmegen, November 2015, a workshop was organized to share experiences of both techniques by several experts in the field and to discuss (long term) outcomes. Specifically, the objective of the meeting was to discuss the main controversies in relation to the technical execution of both procedures in order to make an initial assessment of the limitations of available evidence for clinical decision-making and to formulate a set of preliminary recommendations for current clinical care and future research.

Testicular atrophy following paediatric primary orchidopexy: A prospective study.

BACKGROUND: With the Nordic consensus statement advocating orchidopexy at an earlier age, the present study sought to investigate the outcomes of primary paediatric orchidopexy at a tertiary UK centre. OBJECTIVE: To prospectively assess testicular atrophy following primary orchidopexy for undescended testes in a paediatric population. Secondary outcomes were complication rates and whether outcomes were dependent on grade of operating surgeon. STUDY DESIGN: Prospective data regarding age at operation, classification of the undescended testis, length of follow-up, and subjective comparison of intraoperative and postoperative testicular volumes compared with the contralateral testis were collected. Testicular atrophy was defined as >50% loss of testicular volume or a postoperative testicular volume <25% of the volume of the contralateral testis. Patients were excluded for incomplete data and follow-up <6 months. RESULTS: Data for 234 patients were analysed. Testicular atrophy occurred in 2.6% of cases. There was no reported testicular re-ascent. All secondary acquired cases underwent a previous ipsilateral hernia repair. There was no significant difference in outcomes comparing the grade of surgeon (consultant n = 8, trainee/staff-grade surgeon n = 7-8). There was a trend towards postoperative catch-up growth in approximately one fifth of cases. DISCUSSION: Previous studies have reported a testicular atrophy rate of 5%. The present study reported a similar rate of 2.6%. In agreement with a previous publication, it was also found that testicular atrophy was not dependent on the grade of operating surgeon. The mechanism for testicular catch-up growth is not well understood. Animal studies have supported the hypothesis that increased temperature has a detrimental effect on testicular volume. However, follow-up in the present cohort was short (median 6.9 months), making interpretation of this finding difficult. It is acknowledged that clinical palpation alone to determine testicular volume potentially introduces intra-observer and inter-observer error. However, prospective studies using ultrasound to determine testicular volumes following orchidopexy have reported catch-up growth. CONCLUSION: This study represented one of the larger collections of prospective assessments of outcomes following primary orchidopexy. It was acknowledged that subjectively assessing testicular volume is not ideal; however, the data correlated with similar studies.

Variants of contextual fear conditioning induce differential patterns of Egr-1 activity within the young adult prefrontal cortex.

Contextual fear conditioning is a form of associative learning where animals must experience a context before they can associate it with an aversive stimulus. Single-trial contextual fear conditioning (sCFC) and the context preexposure facilitation effect (CPFE) are two variants of CFC where learning about the context is temporally contiguous (sCFC) with or separated (CPFE) from receiving a footshock in that context. Neural activity within CA1 of the dorsal hippocampus (CA1), amygdala (LA), and prefrontal cortex (PFC) may play a critical role when animals learn to associate a context with a footshock (i.e., training). Previous studies from our lab have found that early-growth-response gene 1 (Egr-1), an immediate early gene, exhibits unique patterns of activity within regions of the PFC following training in sCFC and the CPFE of juvenile rats. In the present study, we extended our studies by examining Egr-1 expression in young adult rats to determine (1) if our previous work reflected changes unique to development or extend into adulthood and (2) to contrast expression profiles between sCFC and the CPFE. Rats that learned context fear with sCFC showed increased Egr-1 in the anterior cingulate, orbitofrontal and infralimbic cortices relative to non-associative controls following training, but expression in prelimbic cortex did not differ between fear conditioned and non-associative controls. In contrast, rats trained in the CPFE also showed increased Egr-1 in all the prefrontal cortex regions, including prelimbic cortex. These findings replicate our previous findings in juveniles and suggest that Egr-1 in specific PFC subregions may be uniquely involved in learning context-fear in the CPFE compared to sCFC.

Refractory depression: mechanisms and evaluation of radically open dialectical behaviour therapy (RO-DBT) [REFRAMED]: protocol for randomised trial.

INTRODUCTION: Only 30-40% of depressed patients treated with medication achieve full remission. Studies that change medication or augment it by psychotherapy achieve only limited benefits, in part because current treatments are not designed for chronic and complex patients. Previous trials have excluded high-risk patients and those with comorbid personality disorder. Radically Open Dialectical Behaviour Therapy (RO-DBT) is a novel, transdiagnostic treatment for disorders of emotional over-control. The REFRAMED trial aims to evaluate the effectiveness and cost-effectiveness of RO-DBT for patients with treatment-resistant depression. METHODS AND ANALYSIS: REFRAMED is a multicentre randomised controlled trial, comparing 7 months of individual and group RO-DBT treatment with treatment as usual (TAU). Our primary outcome measure is depressive symptoms 12 months after randomisation. We shall estimate the cost-effectiveness of RO-DBT by cost per quality-adjusted life year. Causal analyses will explore the mechanisms by which RO-DBT is effective. ETHICS AND DISSEMINATION: The National Research Ethics Service (NRES) Committee South Central - Southampton A first granted ethical approval on 20 June 2011, reference number 11/SC/0146. TRIAL REGISTRATION NUMBER: ISRCTN85784627.

Bioprinting of 3D hydrogels.

Three-dimensional (3D) bioprinting has recently emerged as an extension of 3D material printing, by using biocompatible or cellular components to build structures in an additive, layer-by-layer methodology for encapsulation and culture of cells. These 3D systems allow for cell culture in a suspension for formation of highly organized tissue or controlled spatial orientation of cell environments. The in vitro 3D cellular environments simulate the complexity of an in vivo environment and natural extracellular matrices (ECM). This paper will focus on bioprinting utilizing hydrogels as 3D scaffolds. Hydrogels are advantageous for cell culture as they are highly permeable to cell culture media, nutrients, and waste products generated during metabolic cell processes. They have the ability to be fabricated in customized shapes with various material properties with dimensions at the micron scale. 3D hydrogels are a reliable method for biocompatible 3D printing and have applications in tissue engineering, drug screening, and organ on a chip models.

Applications of three-dimensional (3D) printing for microswimmers and bio-hybrid robotics.

This article will focus on recent reports that have applied three-dimensional (3D) printing for designing millimeter to micrometer architecture for robotic motility. The utilization of 3D printing has rapidly grown in applications for medical prosthetics and scaffolds for organs and tissue, but more recently has been implemented for designing mobile robotics. With an increase in the demand for devices to perform in fragile and confined biological environments, it is crucial to develop new miniaturized, biocompatible 3D systems. Fabrication of materials at different scales with different properties makes 3D printing an ideal system for creating frameworks for small-scale robotics. 3D printing has been applied for the design of externally powered, artificial microswimmers and studying their locomotive capabilities in different fluids. Printed materials have also been incorporated with motile cells for bio-hybrid robots capable of functioning by cell contraction and swimming. These 3D devices offer new methods of robotic motility for biomedical applications requiring miniature structures. Traditional 3D printing methods, where a structure is fabricated in an additive process from a digital design, and non-traditional 3D printing methods, such as lithography and molding, will be discussed.

PD-L1 expression in nonclear-cell renal cell carcinoma.

BACKGROUND: Programmed death ligand-1 (PD-L1) expression in nonclear-cell RCC (non-ccRCC) and its association with clinical outcomes are unknown. METHODS: Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 101 patients with non-ccRCC. PD-L1 expression was evaluated by immunohistochemistry in both tumor cell membrane and tumor-infiltrating mononuclear cells (TIMC). PD-L1 tumor positivity was defined as ≥5% tumor cell membrane staining. For PD-L1 expression in TIMC, a combined score based on the extent of infiltrate and percentage of positive cells was used. Baseline clinico-pathological characteristics and outcome data [time to recurrence (TTR) and overall survival (OS)] were correlated with PD-L1 staining. RESULTS: Among 101 patients, 11 (10.9%) were considered PD-L1+ in tumor cells: 2/36 (5.6%) of chromophobe RCC, 5/50 (10%) of papillary RCC, 3/10 (30%) of Xp11.2 translocation RCC and 1/5 (20%) of collecting duct carcinoma. PD-L1 positivity (PD-L1+) in tumor cells was significantly associated with higher stage (P = 0.01) and grade (P = 0.03), as well as shorter OS (P < 0.001). On the other hand, PD-L1 positivity by TIMC was observed in 57 (56.4%) patients: 13/36 (36.1%) of chromophobe RCC, 30/50 (60%) of papillary RCC, 9/10 (90%) of Xp11.2 translocation RCC and 5/5 (100%) of collecting duct carcinoma. A trend toward shorter OS was observed in patients with PD-L1+ in TIMC (P = 0.08). PD-L1+ in both tumor cell membrane and TIMC cells were associated with shorter TTR (P = 0.02 and P = 0.03, respectively). CONCLUSION: In non-ccRCC, patients with PD-L1+ tumors appear to have worse clinical outcomes, although only PD-L1 positivity in tumor cells is associated with higher tumor stage and grade.