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Programmed death-ligand 1 (PD-L1) copy number gains may be predictive of clinical response to immunotherapy in NSCLC. This study investigated PD-L1 copy number variations in tumor resection and bronchoscopy biopsies and its relationship with PD-L1 tumor cell staining and inflammatory gene expression.PD-L1 gene copy number and mRNA expression were evaluated by real-time polymerase chain reaction in surgically resected NSCLC tumor biopsies (n = 87) and control biopsies (n = 20). A second cohort (n = 15) of bronchoscopy-derived tumor biopsies was analyzed, including multiple biopsies from the same patient across different anatomical sites.PD-L1 mRNA levels strongly correlated with PD-L1 tumor staining (r = 0.55, p0.0001). Interferon-γ mRNA expression associated with PD-L1 immune cell staining, but not PD-L1 tumor cell staining. In contrast, PD-L1 copy number positively associated PD-L1 tumor staining, but not PD-L1 immune cell staining. PD-L1 copy number analysis detected loss (15 of 87 = 17%) and gain (5 of 87 = 7%) of copy number. Tumors with low PD-L1 copy number expressed significantly reduced levels of inflammatory (interferon-γ, interleukin [IL]-6, IL-1β, MMP-9) and immunosuppressive (IL-10, transforming growth factor β) mediators. Analysis of bronchoscopy-derived biopsies revealed low heterogeneity in copy number values across different anatomical sites, in contrast to more variable PD-L1 mRNA expression.Low PD-L1 copy number tumors display reduced PD-L1 expression, reduced PD-L1 tumor cell staining, and an immunologic cold tumor microenvironment. Because PD-L1 copy number values are highly stable across different tumor regions, its evaluation may represent a robust and complimentary biomarker for predicting response to immunotherapy, where low copy number may predict lack of response.

Immune checkpoint inhibitors have become integrated into the clinical management of non-small cell lung cancer (NSCLC). Using RTqPCR, we have previously identified a gene expression panel that detected presence of malignant cells (MMP9:TIMP3 ratio) and quantified PD-L1 transcript levels in small biopsy specimens. However, RTqPCR has diagnostic limitations as it does not generate absolute copy number and is not readily multiplexed. To address this, we have developed a multiplex droplet digital PCR (ddPCR) assay.Biopsies obtained from NSCLC patients (n = 48 adenocarcinoma and n = 40 squamous cell carcinoma) and control lung biopsy specimens (n = 20) were analysed. Absolute MMP9, TIMP3 and PD-L1 transcript copy numbers were determined within a single assay by multiplex ddPCR using Taqman primers and the QX200 Droplet Digital PCR System.Using our optimised triplex ddPCR assay, the MMP9:TIMP3 ratio was significantly elevated in NSCLC biopsies and using a cut-off of0.028, was 99% (95% CI; 80.5-94.5) sensitive and 80% specific for identifying malignant biopsies. The PD-L1:TIMP3 ratio significantly associated with PD-L1 tumour cell immunohistochemistry staining (r = 0.539, p 0.0001) and was significantly higher in biopsies with50% PD-L1 tumour cell staining (p 0.0001). In summary, a major advantage of our workflow is that it can accurately quantify PD-L1 tumour levels and provide sufficient nucleic acid for screening additional targetable mutations such as EGFR, ALK and ROS1 from a single small biopsy, thereby potentially avoiding the need for re-biopsy. Future studies will need to determine diagnostic ddPCR values that are predictive of clinical response to PD-1/PD-L1 immunotherapy.

Objective The feasibility and effectiveness of concomitant use of transcranial direct current stimulation (tDCS) with electroconvulsive therapy (ECT) has not been investigated. The study principally aimed at determining whether tDCS when combined with ECT improved the speed of antidepressant response. Secondarily, the ease of generation of seizures during electroconvulsive therapy and cognitive outcomes were investigated. Methods Consecutive patients referred for ECT to treat major depression were randomized to tDCS with dorsolateral prefrontal electrode placements (n = 8) or sham (n = 8) used daily and just before thrice weekly, 6 times threshold, right unilateral ultrabrief (0.3 ms) pulse width ECT. Change of depression severity was determined using the Montgomery Asberg Depression Rating Scale along with cognitive assessments using Montreal Cognitive Assessment and visual memory testing at weeks 1 and 2, which were compared with baseline. Results Change of depression severity from baseline was similar in tDCS and ECT compared with sham tDCS and ECT at week1 (mean [standard deviation {SD}] = 16.00 [6.78]; 13.75 [7.83]; P = 0.89) and at week 2 (mean [SD] = 23.00 [4.96]; 19.75 [9.85], P = 0.08). No between-group differences were obtained in the cognitive tests at weeks 1 and 2. Combining tDCS with ECT resulted in higher restimulation: 62.5% requiring 3 stimulations to achieve threshold in contrast to 12.5% with sham tDCS and ECT (P = 0.04). The mean suprathreshold dose was higher in the tDCS and ECT group compared with sham tDCS and ECT: mean [SD] = 144.0 [43.54] and mean [SD] = 122.4 [20.36], P = 0.04, respectively. Conclusions Concomitant use of tDCS with ultrabrief right unilateral ECT is feasible and safe albeit with higher rates of restimulation when tDCS was combined with ECT. However, there were no statistically significant differences in the speed of antidepressant response or cognitive outcomes at weeks 1 and 2 after the commencement of treatments.

Immunotherapy has transformed treatment of advanced non-small-cell lung cancer (NSCLC) patients leading to remarkable long-term survival benefit. However, only about 20% of advanced NSCLC patients typically respond to immune checkpoint inhibitors (ICIs) that target the PD-1/PD-L1 pathway. The only validated biomarker for ICI therapy is the PD-L1 immunohistochemistry (IHC) test, which is considered an imperfect assay due to several variables including availability and integrity of tumour tissue, variability in staining/scoring techniques and heterogeneity in PD-L1 protein expression within and across tumour biopsies. Herein, we discuss integrating minimally invasive EBUS bronchoscopy procedures with novel molecular approaches to improve accuracy and sensitivity of PD-L1 testing. EBUS guided bronchoscopy facilitates repeated sampling of tumour tissue to increase the probability of detecting PD-L1 positive tumours. Since intra-tumoural PD-L1 (CD274) copy number is reported to be less heterogeneous than PD-L1 protein detection, quantifying PD-L1 transcript levels may increase detection of PD-L1 positive tumours. PD-L1 transcript levels show excellent concordance with PD-L1 IHC scoring and multiplex digital droplet PCR (ddPCR) assays that quantify absolute PD-L1 transcript copy number have been developed. ddPCR can also be automated for high throughput detection of low abundant variants with excellent sensitivity and accuracy to improve the broader application of diagnostic cut-off values. Optimizing diagnostic workflows that integrate optimal EBUS bronchoscopy procedures with emerging molecular ICI biomarker assays may improve the selection criteria for ICI therapy benefit.

Aim Cryptorchidism affects 2%–4% of newborn boys and causes infertility and cancer. While normal androgen function is required for successful inguinoscrotal descent, its exact role on gubernacular morphology remains unidentified. We aimed to decipher the effect of androgen blockade on the gubernaculum and surrounding structures. Methods Genetically modified mice with androgen receptor knock out (ARKO) were sectioned at ages E17, D0, and D2 for immunohistochemical analysis and D4 for morphological analysis (with ethical approval; A644). Mutants and control littermates were labeled with Ki67, Desmin, and Pax7 to identify the degree of gubernaculuar eversion and the composition of the growth center in the gubernaculum, using light or confocal microscopy. Results Androgen blockade prevented gubernacular eversion in all animals aged between E17 and D2 when compared to wild types. Furthermore, a growth center was visible, as indicated by a ‘swirl’ of immature fibroblasts, in D2 animals but was absent in ARKO mice. Moreover, the gubernacular cord was seen to increase in ARKO mice when compared to wild types and increased in size with age. There were no labeling differences in the antibodies tested for gubernacular differentiation. Conclusion Gubernacular eversion in rodents prior to inguinoscrotal migration was androgen dependent, as well as maintenance of gubernacular cord length. This study shows that androgen blockade causes cryptorchidism in mice by preventing gubernacular eversion and possibly by preventing shortening of the gubernacular cord. Altering the morphology of the gubernaculum in response to androgen clearly contributes to the clinical problem of cryptorchidism.

Simple Summary Aspirin-triggered resolvin D1 (AT-RvD1) is biosynthesised by leukocytes as a mechanism to resolve inflammation during infection and/or injury. Emerging studies reveal that AT-RvD1 also has anti-cancer properties associated with stimulating macrophage-mediated clearance of tumour debris. No study to date has investigated how AT-RvD1 influences the neutrophil to lymphocyte ratio (NLR) in lung cancer, an established marker of poor prognosis. The biosynthesis of AT-RvD1 is dependent on the ALOX5 gene, and we reveal that ALOX5 mRNA expression was markedly reduced in lung adenocarcinoma tumours. We next utilised an oncogenic KrasG12D lung adenocarcinoma mouse model to investigate the efficacy of AT-RvD1 in vivo. We show for the first time that AT-RvD1 reduces tumour growth in the lungs of KrasG12D mice and alters the immune landscape in tumours by reducing the NLR. Abstract Tumour-associated neutrophils (TANs) can support tumour growth by suppressing cytotoxic lymphocytes. AT-RvD1 is an eicosanoid that can antagonise neutrophil trafficking instigated by ALX/FPR2 ligands such as serum amyloid A (SAA). We aimed to establish whether SAA and ALOX5 expression associates with TANs and investigate the immunomodulatory actions of AT-RvD1 in vivo. MPO-positive neutrophils were quantified in tumour blocks from lung adenocarcinoma (n = 48) and control tissue (n = 20) by IHC. Tumour expression of SAA and ALOX5 were analysed by RTqPCR and an oncogenic KrasG12D lung adenocarcinoma mouse model was used to investigate the in vivo efficacy of AT-RvD1 treatment. ALOX5 expression was markedly reduced in lung adenocarcinoma tumours. The SAA/ALOX5 ratio strongly correlated with TANs and was significantly increased in tumours harbouring an oncogenic KRAS mutation. AT-RvD1 treatment reduced tumour growth in KrasG12D mice, which was accompanied by suppressed cellular proliferation within parenchymal lesions. In addition, AT-RvD1 significantly reduced the neutrophil to lymphocyte ratio (NLR), an established prognostic marker of poor survival in adenocarcinoma. This study identifies a novel molecular signature whereby elevated levels of SAA relative to ALOX5 favour accumulation of TANs. Furthermore, the ALOX5/5-LO enzymatic product, AT-RvD1, markedly reduced the NLR and suppressed tumour growth in KrasG12D mice.

Matrix metalloproteinase-9 (MMP-9) is increased in a number of pathological lung conditions, where the proteinase contributes to deleterious remodelling of the airways. While both lung cancer and COPD are associated with increased MMP-9 expression, the cellular and molecular drivers of MMP-9 remain unresolved. In this study, MMP-9 transcript measured within the tumour region from patients with non-small-cell lung cancer (NSCLC) and coexisting COPD was found to be uniformly increased relative to adjacent tumour-free tissue. MMP-9 gene expression and immunohistochemistry identified tumour-associated neutrophils, but not macrophages, as a predominant source of this proteinase. In addition, PTEN gene expression was significantly reduced in tumour and there was evidence of epithelial MMP-9 expression. To explore whether PTEN can regulate epithelial MMP-9 expression, a small interfering (si)RNA knockdown strategy was used in Beas-2B bronchial epithelial cells. PTEN knockdown by siRNA selectively increased MMP-9 expression in response to lipopolysaccharide in a corticosteroid-insensitive manner. In summary, tumour-associated neutrophils represent an important source of MMP-9 in NSCLC, and loss of epithelial PTEN may further augment steroid-insensitive expression.

Background/aim In cryptorchidism perinatal failure to switch off Oct4, a germ cell (GC) marker, may lead to carcinoma in situ . We aimed to analyze Oct4 expression during mouse gonocyte transformation into spermatogonial stem cells (SSC). Materials and methods Testes from OG2 (Oct4-promoter driven e GFP) mice at embryonic day (E) 17 and postnatal day P0–10 underwent immunohistochemistry and immunoblotting. Antibodies against MVH, AMH, Ki67, and c-Kit were visualized by confocal microscopy. Numbers of Oct4-GFP + GC and Oct4-GFP − GC/tubule were counted using ImageJ. Data were analyzed using nonparametric one-way ANOVA. Results GC from E17-P4 were Oct4-GFP + . Numbers of Oct4-GFP − GC/tubule increased from P6–10, whereas Oct4-GFP + GC/tubule numbers remained similar between P6 and P10. Sertoli cells proliferated from E17–P10, whereas GC only proliferated from P2. Gonocytes (Oct4-GFP + /c-Kit − ) central in tubules migrated to the basement membrane to become prospermatogonia (Oct4-GFP + /c-Kit − ) and then SSC (Oct4-GFP + /c-Kit + ) from day 4 and further developed into Oct4-GFP − /c-Kit + at P6. Conclusion In Oct4-GFP mice both centrally located gonocytes and prospermatogonia located at the tubular basement membrane were Oct4-GFP + /c-Kit − before further developing into SSC (Oct4-GFP + /c-Kit + ). This indicates that Oct4 is important in gonocyte transformation into SSC. Understanding this process will aid GC tumor diagnostics and fertility potential in boys with UDT undergoing orchidopexy.

Multiple biomarkers are under evaluation to guide the use of immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC), including programed death ligand 1 (PD-L1) tumor cell staining. We have developed a new approach that accurately quantifies PD-L1 status and identifies multiple mutations by using a single bronchoscopy specimen. A novel molecular marker was identified to detect the presence of malignant cells in radial endobronchial ultrasound bronchial brushings from NSCLC (n = 15) and benign (n = 13) nodules by quantitative real-time RT-PCR (RT-qPCR). The MMP9:TIMP3 transcript ratio was significantly increased in NSCLC and using receiver operating characteristic curve analysis accurately discriminated malignant and benign bronchoscopy specimens (area under the curve = 0.98; 95% CI, 0.93-1; P 0.0001). Utilizing the same specimens, PD-L1 expression and multiple oncogenic mutations were detected by RT-qPCR and next-generation sequencing. A second archive of snap-frozen squamous cell carcinoma (n = 40) and control (n = 20) biopsies with matching formalin-fixed, paraffin-embedded slides were used to compare PD-L1 status by immunohistochemistry and RT-qPCR. The biopsy cohort confirmed that the MMP-9:TIMP3 ratio was predictive of malignancy and demonstrated that PD-L1 transcript expression was concordant with PD-L1 tumor cell membrane staining in NSCLC (Spearman r = 0.636, P 0.0001). This rapid molecular approach can detect malignant cells and using the same single bronchoscopy specimen can generate high-quality unfixed nucleic acid that accurately quantify PD-L1 status and identify multiple oncogenic mutations.

Background/Aim It has been proposed that androgens control inguinoscrotal testicular descent via release of calcitonin gene-related peptide (CGRP) from a masculinised genitofemoral nerve (GFN). As there are androgen receptors in the inguinoscrotal fat pad (IFP) during the window of androgen sensitivity (E14-17 in mouse embryos), we tested the hypothesis that neurotrophins in the IFP may masculinise the sensory fibers of the GFN supplying the gubernaculum and IFP prior to gubernacular migration. Methods Androgen-receptor knockout (ARKO) and wild-type (WT) mouse embryos were collected at E17, with ethical approval (AEC 734). Sagittal sections of IFP, mammary area and bulbocavernosus (BC) muscle were processed for standard histology and fluorescent immunohistochemistry for ciliary neurotrophic factor (CNTF), ciliary neurotrophic factor receptor (CNTFR) and cell nuclei (DAPI). Results In the ARKO mouse CNTFR immunoreactivity (CNTFR-IR) was increased in the IFP but decreased in BC. Perinuclear staining of CNTF-IR was seen in mouse sciatic nerve but only weakly in IFP. In the mammary area, also supplied by GFN, there were no differences in IR staining. Conclusion This study found CNTFR-IR in the IFP was negatively regulated by androgen, suggesting that CNTF signaling may be suppressed in GFN sensory nerves to enable CGRP expression for regulating gubernacular migration in the male, but not the female. The indirect action of androgen via the GFN required for testicular descent may be one of the sites of anomalies in the putative multifactorial cause of cryptorchidism.

As the transient postnatal hormone surge in humans, known as ‘minipuberty’, occurs simultaneously with key steps in germ-cell development, we investigated whether similar changes occur in the hypothalamic–pituitary–testicular axis of neonatal mice at a time that would coincide with gonocyte transformation into spermatogonial stem cells (SSC). Serum and testes were collected from C57Bl/6 mice at embryonic Day 17 (E17), birth (postnatal Day 0; P0) and daily until P10. Serum FSH and testosterone levels in both serum and testes were analysed and gene expression of FSH receptor (Fshr), luteinising hormone receptor (Lhr), anti-Müllerian hormone (Amh), octamer-binding transcription factor 4 (Oct-4), membrane type 1 metalloprotease (Mt1-mmp), proto-oncogene C-kit and promyelocytic leukaemia zinc finger (Plzf ) was quantified by real-time polymerase chain reaction. We found a transient surge of serum and testicular testosterone levels between P1 and P3 and a gradual increase in FSH from P1 to P10. Testis Lhr expression remained low from P0 until P10 but Fshr expression peaked between P3 and P6 (P

Purpose: Undescended testis leads to infertility and malignancy resulting from aberrant germ cell development. Androgens are proposed to control early germ cell development during the transient postnatal surge of gonadotropins and androgen, known as mini-puberty. We assessed the effect of androgen receptor on perinatal germ cell development in mice.Materials and Methods: Testes from androgen receptor knockout mice and wildtype littermates (3 to 4 per group) were collected at embryonic day 17 and postnatal days 0 (birth), 2, 4, 6, 8 and 10 for immunohistochemical analysis. Antibodies against mouse VASA homologue (germ cell marker), antimullerian hormone (Sertoli cell marker), Ki67 (proliferating cell marker) and DAPI (nuclei) were used and visualized by confocal microscopy. Number of germ cells per tubule, germ cells on the tubular basement membrane and Sertoli cells per tubule, and percentage of proliferating germ cells (Ki67(+)) per tubule and germ cells (Ki67(+)) on the basement membrane on confocal images were counted using Image J, version 1.44 (http://imagej.nih.gov/ij/). Data were analyzed using nonparametric one-way ANOVA with GraphPad Prism (R) 5.02 software.Results: In wild-type and androgen receptor knockout testes germ cells per tubule decreased from embryonic day 17 to postnatal day 2, then increased normally. Number of mouse VASA homologue positive germ cells per tubule and germ cells on the basement membrane were similar in androgen receptor knockout and wild-type testes (p > 0.05) at each age, and percentages of proliferating germ cells (Ki67(+)) per tubule and proliferating germ cells on the basement membrane were similar at each age (p > 0.05).Conclusions: Androgen receptors are not required for gonocyte migration from the center of the testicular tubules to the basement membrane and transformation into spermatogonia stem cells up to day 10 in androgen receptor knockout mice. Identifying nonandrogenic factors might improve the fertility potential of boys with undescended testis who are undergoing orchiopexy.

A new radiotracer, 99mTc-imidodiphosphate, has been investigated in a series of 101 consecutive admissions to a Coronary Care Unit in a large teaching hospital to assess its potential as an acute myocardial infarct imaging agent. This agent shows high sensitivity for transmural infarction (0.96), subendocardial infarction (0.88) and high overall specificity (1.00). The clarity of the images produced, and the time after the acute episode when studies were positive suggest that it is the agent of choice for imaging the acutely infarcted myocardium.

Clinico-anatomic correlation of the findings in twenty-three cases of ruptured chordae tendineae indicated that rupture associated with other disease of the mitral valve apparatus (group I) differs greatly from isolated rupture of chordae tendineae (group IIA), which presents a distinct clinical-pathologic syndrome. In six additional cases a clinical diagnosis of this syndrome was made (group IIB). Group I represents the end result of a variety of conditions: rheumatic fever, bacterial endocarditis, congenital mitral valve disease and postinfarctional papillary muscle dysfunction. In group IIA no major abnormality of the heart was found and no clinical or pathologic clues to suggest an etiologic factor for rupture of the chordae were detected. The characteristic clinical picture of this syndrome is the result of (1) preferential involvement of the chordae supporting the posterior leaflet of the mitral valve, and (2) the sudden development of mitral valve incompetence. Loss of chordal support by a portion of the posterior leaflet during systole produces a hood-like prolapse of the unsupported portion into the left atrium causing a high velocity jet to be directed toward the upper part of the atrial septum adjacent to the aortic root. This jet creates vibrations heard as a systolic murmur and often felt as a systolic thrill in the "aortic" area of the right upper thorax—a finding closely imitating aortic stenosis. This is the most specific clinical feature of the syndrome of isolated rupture of the chordae tendineae. The sudden appearance of mitral valve incompetence often leads to abrupt development of cardiac failure without the production of gross cardiomegaly, severe left atrial enlargement or atrial fibrillation. This sudden development is not specific for isolated rupture but occurs in other forms of mitral insufficiency, notably after bacterial endocarditis. Clinical identification of this syndrome has practical significance because of the availability of low-risk corrective valvuloplasty, which has been shown to produce satisfactory and lasting results. Analysis of twenty cases of isolated rupture of the chordae tendineae reveals that contrary to common belief, rupture of chordae tendineae is not necessarily a serious cardiac event, as milder forms (presumably due to rupture of a single chorda) are consistent with a fully compensated, asymptomatic state for many years, perhaps even permanently.

Extraskeletal uptake of tracer, presumably in the lymph nodes, is an uncommon finding in bone scans. It has been seen only when there is extravasation of tracer at the injection site. The phenomenon appears to be the result of colloid formation.