Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome.

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

High-Throughput Simultaneous mRNA Profiling Using nCounter Technology Demonstrates That Extracellular Vesicles Contain Different mRNA Transcripts Than Their Parental Prostate Cancer Cells.

Extracellular vesicles (EVs) are nano-sized lipid bilayer encapsulated particles with a molecular cargo that appears to play important roles within the human body, such as in cell-to-cell communication. Unraveling the composition of EV cargos remains one of the most fundamental steps toward understanding the role of EVs in intercellular communication and the discovery of new biomarkers. One of the unmet needs in this field is the lack of a robust, sensitive, and multiplexed method for EV mRNA profiling. We established a new protocol using the NanoString low RNA input nCounter assay by which the targeted mRNA transcripts in EVs can be efficiently and specifically amplified and then assayed for 770 mRNAs in one reaction. Prostate cancer cells with epithelial (PC3-Epi) or mesenchymal (PC3-EMT) phenotypes and their progeny EVs were analyzed by the same panel. Among these mRNAs, 157 were detected in PC3-Epi EVs and 564 were detected in PC3-EMT EVs. NOTCH1 was the most significantly abundant mRNA transcripts in PC3-EMT EVs compared to PC3-Epi EVs. Our results demonstrated that when cells undergo epithelial-to-mesenchymal transition (EMT), a more active loading of cancer progression-related mRNA transcripts may occur. The mRNA cargos of EVs derived from mesenchymal prostate cancer cells may contribute to the pro-EMT function. We found that mRNA transcripts are different in progeny EVs compared to parental cells. EV cargos are not completely reflective of their cell origin, and the underlying mechanism of cargo sorting is complicated and needs to be further elucidated.

Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform.

BACKGROUND: With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids. METHODS: Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes. RESULTS: Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease. CONCLUSIONS: The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

Neurodevelopmental Outcomes after Intravitreal Bevacizumab Therapy for Retinopathy of Prematurity: A Prospective Case-Control Study.

PURPOSE: To evaluate the neurodevelopmental and ocular developmental outcomes in premature children who have undergone intravitreal bevacizumab injection (IVB) for treatment of type 1 retinopathy of prematurity (ROP). DESIGN: Prospective case-control study. PARTICIPANTS: We enrolled 3 groups of premature patients: premature children who had no history of ROP (group 0), premature children with history of ROP without treatment (group 1), and premature children with ROP who had received a single IVB (0.625 mg; group 2). METHODS: Ocular developmental assessment, including cycloplegic refractometry, axial length, Cardiff acuity, and neurodevelopmental assessment via the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley III), were performed at 1 to 3 years of age and were compared between groups. MAIN OUTCOME MEASURES: Ocular developmental outcomes and Bayley III scores. RESULTS: A total of 148 patients (85 boys and 63 girls) were included. The mean age at assessment was 1.49±0.59 years. Group 0 patients demonstrated significantly higher gestational age (GA), birth weight, and Apgar scores compared with group 1 and 2 patients. There were no significant differences between groups 1 and 2 in demographics or systemic risk factors except for lower GA in group 2. The cylindrical power was significantly larger in groups 1 and 2 compared with group 0. The spherical equivalent was significantly more myopic and the Cardiff acuity was significantly poorer in group 2 than in group 0. There were no significant differences between groups 1 and 2 in refractive status, axial length, or Cardiff acuity. Neurodevelopmental assessment using Bayley III showed no significant difference among the 3 groups in any aspect after adjusting for GA and other systemic risk factors. The risks for poor neurodevelopmental outcomes also were not significantly different. CONCLUSIONS: At the mean age of 1.5 years, children with prior history of IVB (group 2) showed similar refractive and visual outcomes and similar neurodevelopmental outcomes compared with premature patients with ROP without requirement of treatment (group 1), although there is a possibility that a small but clinically significant difference may not have been detected in the current study.

Changes in systemic vascular endothelial growth factor levels after intravitreal injection of aflibercept in infants with retinopathy of prematurity.

BACKGROUND: To investigate the levels of VEGF in the systemic circulation of patients with type 1 ROP who received intravitreal injections of 1 mg (0.025 mL) aflibercept (IVA) or 0.625 mg (0.025 mL) bevacizumab (IVB). METHODS: Patients who had type 1 ROP and received either IVA or IVB were enrolled in this prospective study. Serum and plasma samples were collected prior to and up to 12 weeks after IVB or IVA treatment. The serum and plasma VEGF levels were measured using enzyme-linked immunosorbent assays (ELISAs), and the platelet levels in the blood were also quantified. The serum and plasma levels of VEGF, as well as the ratio of VEGF to platelet count (VEGF/PLT) were measured prior to and up to 12 weeks after anti-VEGF treatment. RESULTS: In total, 14 patients with type 1 ROP were enrolled in this study; five patients received IVA, and nine patients received IVB. Following either IVA or IVB treatment, all the eyes (100%) showed complete resolution of ROP-induced abnormal neovascularization and presented continued vascularization toward the peripheral retina. Compared to baseline, the serum VEGF levels were significantly reduced in the ROP patients up to 12 weeks after either IVA or IVB treatments (all P < 0.05). At 2, 4, and 8 weeks after intravitreal injection, the serum VEGF levels were more suppressed in the IVB group than in the IVA group (P = 0.039, P = 0.004, and P = 0.003, respectively). The serum VEGF/PLT ratio after IVA or IVB showed similar reductions and trends as the serum VEGF data. Changes in the plasma VEGF levels could not be properly assessed because some of the samples had VEGF levels below the detection limit of the ELISA. CONCLUSIONS: Serum VEGF levels and the VEGF/PLT ratio in patients with type 1 ROP were suppressed for 3 months after treatment with either IVA or IVB, but the suppression of systemic VEGF was more pronounced in patients treated with IVB than those treated with IVA.

Corneal Endothelial Morphology and Ocular Biometric Indexes in Premature Children With and Without Retinopathy of Prematurity.

Purpose: The purpose of this study was to analyze human corneal endothelial cells (HCECs) morphology and ocular biometrics in premature (PM) children with or without retinopathy of prematurity (ROP). Methods: Retrospective data on patient demographics, HCECs status, and ocular biometrics with at least 2 visits between 2016 and 2021 were reviewed. The main outcomes were endothelial cell density (ECD), coefficient of variation (CV), hexagonal cell ratio (HEX), central corneal thickness (CCT), axial length, anterior chamber depth, keratometry, corneal diameter, pupil diameter, and refraction status. Generalized estimating equation was used to evaluate the differences between PM no-ROP and ROP groups. We also analyzed the trend of ECD, CV, HEX, and CCT change with age between groups. Results: The study included 173 PM patients without ROP and 139 patients with ROP. A total of 666 and 544 measurements were recorded in the PM no-ROP and ROP groups, respectively. The ROP group had higher spherical power, myopic spherical equivalent (SE), and steeper steep keratometry (K; P < 0.05). The ROP group had higher CV (P = 0.0144), lower HEX (P = 0.0012) and thicker CCT (P = 0.0035). In the HCECs parameters, the ROP group had slower ECD decrement (P < 0.0001), faster CV decrement (P = 0.0060), and faster HEX increment (P = 0.0001). A difference in corneal morphology changes between the ROP and PM no-ROP groups were prominent in patients with lower gestational age (GA) in the subgroup analysis. Conclusions: Worse HCECs morphology and higher myopic status were initially observed in patients with prior ROP but not in PM patients with no-ROP. ECD and HCECs morphology improved with age, especially in patients with low GA.

The state of the human proteome in 2012 as viewed through PeptideAtlas.

The Human Proteome Project was launched in September 2010 with the goal of characterizing at least one protein product from each protein-coding gene. Here we assess how much of the proteome has been detected to date via tandem mass spectrometry by analyzing PeptideAtlas, a compendium of human derived LC-MS/MS proteomics data from many laboratories around the world. All data sets are processed with a consistent set of parameters using the Trans-Proteomic Pipeline and subjected to a 1% protein FDR filter before inclusion in PeptideAtlas. Therefore, PeptideAtlas contains only high confidence protein identifications. To increase proteome coverage, we explored new comprehensive public data sources for data likely to add new proteins to the Human PeptideAtlas. We then folded these data into a Human PeptideAtlas 2012 build and mapped it to Swiss-Prot, a protein sequence database curated to contain one entry per human protein coding gene. We find that this latest PeptideAtlas build includes at least one peptide for each of ~12500 Swiss-Prot entries, leaving ~7500 gene products yet to be confidently cataloged. We characterize these "PA-unseen" proteins in terms of tissue localization, transcript abundance, and Gene Ontology enrichment, and propose reasons for their absence from PeptideAtlas and strategies for detecting them in the future.

Changes in anisometropia by age in children with hyperopia, myopia, and antimetropia.

Anisometropia is a unique condition of both eyes and it is associated with vision problems such as amblyopia and reduced stereoacuity. Previous studies have not reported its change pattern by age and its correlation with the refractive condition of both eyes. This study aims to compare the changes in anisometropia by age in children with hyperopia, myopia, and antimetropia. In total, 156 children were included. Children aged 3-11 years with anisometropia ≥ 1.00 D were followed up for ≥ 1 year with ≥ 2 visits at two medical centers in Taiwan. Refractive errors by cycloplegic autorefractometry, best-corrected visual acuity, eye position, and atropine use were recorded. The children were divided into hyperopic, myopic, and antimetropic groups. The results showed that anisometropia decreased in children aged < 6 years (3.34-2.96 D; P = 0.038) and increased in older children (2.16-2.55 D; P = 0.005). In children aged 3, 4, 5, and 6 years, the mean anisometropia was higher in children with myopia and antimetropia than in those with hyperopia (P = 0.005, 0.002, 0.001, and 0.011, respectively). The differences were not significant in children aged > 6 years (all P > 0.05). The factors associated with changes in anisometropia were age, refractive group, amblyopia, and strabismus. Anisometropia decreased with age in children younger than 6 years, and the changes in anisometropia was found in children with myopia and antimetropia.

Visual acuity, optical components, and macular abnormalities in patients with a history of retinopathy of prematurity.

PURPOSE: To examine the optical components and spectral-domain optical coherence tomography (OCT) findings in children with a history of retinopathy of prematurity (ROP) and to identify any associations between the OCT findings and the visual acuities of the patients. DESIGN: Prospective, case-controlled study. PARTICIPANTS AND CONTROLS: Children who were between 6 and 14 years of age were divided into the following 4 groups: Patients with a history of threshold ROP who had been treated using laser therapy or cryotherapy (group 1), patients with regressed ROP who had not received any treatment (group 2), patients who were born prematurely but who had no history of ROP (group 3), and normal full-term children (group 4). The posterior poles of the eyes of all of the patients seemed to be normal. METHODS: Visual acuities, optical components, and macular thicknesses were measured in 4 groups of patients, and comparisons between the groups were made. Macular thicknesses were measured using OCT. MAIN OUTCOME MEASURES: Visual acuity (VA), optical components, and OCT findings. RESULTS: We enrolled 133 patients in the study. Patients in group 1 had significantly thicker foveas than the other patients, as demonstrated by OCT, and this finding was negatively correlated with gestational age. The incidence of abnormal foveal contours among patients in group 1 was significantly higher than among the rest of the patients. Retention of the inner retinal layers was noted in group 1 patients; however, the structure of the outer retina remained intact. Greater degrees of myopic shift and astigmatism, steeper corneal curvatures, shallower anterior chamber depths, and thicker lenses were noted in previously treated ROP patients. These findings corresponded with poor VA and high refractive errors in group 1 patients. CONCLUSIONS: Patients with a history of threshold ROP are more likely to show abnormal foveal development and have a poorer visual prognosis than other patient groups despite a fundus with no macular dragging, disc dragging, or retinal detachment. A steeper corneal curvature, shallower anterior chamber, and greater lens thickness are the main changes in the optical components in these patients.

RNA profiling of circulating tumor cells systemically captured from diagnostic leukapheresis products in prostate cancer patients.

The application of circulating tumor cells (CTCs) in both clinical practice and research has been continuously limited by the rare number of targets that can be found in a tube of peripheral blood. Diagnostic leukapheresis (DLA) was used to increase the sampling volume. AdnaTest was used to process the whole leukopak, and the RNAs of captured CTCs was then profiled by NanoString nCounter platform. Spike-in experiments and leukopaks from patients with metastatic prostate cancer were used to validate this new strategy. The whole leukopak was further concentrated five times to reduce the total volume from 150 â€‹mL to 30 â€‹mL, which enabled it to be processed by 3 separate AdnaTest kits. The spike-in experiment demonstrated a reliable capture when there were more than 100 cancer cells/10 â€‹mL of concentrated leukopak. In 1 out of 5 real patient samples, CTCs were only detected in the leukopak, but not in peripheral blood. The RNA profiling of DLA CTCs indicated a more aggressive phenotype of CTCs occurred when the patient was experiencing a disease relapse, even when the serum prostate specific antigen (PSA) level was still relatively low and CTCs in peripheral blood were not detectable. We established a new protocol, integrating DLA, AdnaTest and NanoString nCounter technology, to profile RNAs from CTCs captured from a large blood screening volume. The new protocol can process the whole leukopak with sensitive CTC capture. The RNA profiling of CTCs can provide valuable information for disease monitoring.

Digital multiplexed analysis of circular RNAs in FFPE and fresh non-small cell lung cancer specimens.

Although many studies highlight the implication of circular RNAs (circRNAs) in carcinogenesis and tumor progression, their potential as cancer biomarkers has not yet been fully explored in the clinic due to the limitations of current quantification methods. Here, we report the use of the nCounter platform as a valid technology for the analysis of circRNA expression patterns in non-small cell lung cancer (NSCLC) specimens. Under this context, our custom-made circRNA panel was able to detect circRNA expression both in NSCLC cells and formalin-fixed paraffin-embedded (FFPE) tissues. CircFUT8 was overexpressed in NSCLC, contrasting with circEPB41L2, circBNC2, and circSOX13 downregulation even at the early stages of the disease. Machine learning (ML) approaches from different paradigms allowed discrimination of NSCLC from nontumor controls (NTCs) with an 8-circRNA signature. An additional 4-circRNA signature was able to classify early-stage NSCLC samples from NTC, reaching a maximum area under the ROC curve (AUC) of 0.981. Our results not only present two circRNA signatures with diagnosis potential but also introduce nCounter processing following ML as a feasible protocol for the study and development of circRNA signatures for NSCLC.

Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform.

Extracellular vesicles (EVs) are double-layered phospholipid membrane vesicles that are released by most cells and can mediate intercellular communication through their RNA cargo. In this study, we tested if the NanoString nCounter platform can be used for the analysis of EV-mRNA. We developed and optimized a methodology for EV enrichment, EV-RNA extraction and nCounter analysis. Then, we demonstrated the validity of our workflow by analyzing EV-RNA profiles from the plasma of 19 cancer patients and 10 controls and developing a gene signature to differentiate cancer versus control samples. TRI reagent outperformed automated RNA extraction and, although lower plasma input is feasible, 500 µL provided highest total counts and number of transcripts detected. A 10-cycle pre-amplification followed by DNase treatment yielded reproducible mRNA target detection. However, appropriate probe design to prevent genomic DNA binding is preferred. A gene signature, created using a bioinformatic algorithm, was able to distinguish between control and cancer EV-mRNA profiles with an area under the ROC curve of 0.99. Hence, the nCounter platform can be used to detect mRNA targets and develop gene signatures from plasma-derived EVs.

MicroRNA-126 inhibits pathological retinal neovascularization via suppressing vascular endothelial growth factor expression in a rat model of retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) is the principal growth factor responsible for the retinal neovascularization in the pathogenesis of retinopathy of prematurity (ROP). Current therapies for ROP include laser ablation and intravitreal anti-VEGF injection. However, these treatments either destroy the peripheral retina or associate with problems of persistent peripheral avascular retina or later recurrence of ROP. In the present study we investigated a new therapeutic approach by exploring the potential role of a specific microRNA, miR-126, in regulating VEGFA expression and retinal neovascularization in a rat oxygen-induced retinopathy (OIR) model. We demonstrated that miR-126 mimic and plasmid effectively suppresses VEGFA mRNA expression in both human and rat retinal pigment epithelium cell lines, quantified with qRT-PCR. Animal experiments on rat OIR model revealed that intravitreal injection of miR-126 plasmid efficiently downregulated VEGFA expression in the intraocular fluid and retinal tissues measured by ELISA, and significantly suppressed retinal neovascularization, which was confirmed by calculating sizes of neovascularization areas on fluorescence microscopic images of flat mounted retina stained with Alexa Fluor 594-conjugated isolectin B4 to visualize blood vessels. Together, these results showed that intravitreal injection of miR-126 plasmid could inhibit retinal neovascularization by down-regulating VEGFA expression, suggesting a potential therapeutic effect for ROP.

Myopia in school-aged children with preterm birth: the roles of time spent outdoors and serum vitamin D.

AIMS: To analyse the factors associated with myopia in school-aged children with preterm birth and with or without retinopathy of prematurity (ROP). METHODS: Children born prematurely between January 2010 and December 2011 were enrolled in this cross-sectional study when they reached school age between April 2017 and June 2018 in a referral centre. The main parameters were cycloplegic refraction, time spent outdoors and serum 25-hydroxyvitamin D (25(OH)D) concentration. RESULTS: A total of 99 eyes from 99 children with a mean age of 6.8 years underwent analysis. The average time spent outdoors was significantly higher in the non-myopic group (0.9 ± 0.5 hours/day) than in the myopic group (0.7 ± 0.3 hours/day) (p = 0.032). After adjustment for age, sex, number of myopic parents, ROP severity, near-work time and serum 25(OH)D concentration, more time spent outdoors was correlated with a lower odds of myopia (OR, 0.13 per additional hour per day; 95% CI, 0.02-0.98; p = 0.048). Mean serum 25(OH)D concentrations were similar between the myopic and non-myopic groups (49.7 ± 13.6 and 48.8 ± 14.0 nmol/mL; p = 0.806) and were not correlated with spherical equivalence power (r = -0.09; p = 0.418). Vitamin D insufficiency was present in 57% of the participants. CONCLUSIONS: Among preterm children with or without ROP, more time spent outdoors was associated with lower odds of myopia. The serum 25(OH)D concentration was not associated with myopia, but a high proportion of the participants had insufficient levels.

CCL2 is induced by chemotherapy and protects prostate cancer cells from docetaxel-induced cytotoxicity.

BACKGROUND: Metastatic prostate cancer is either inherently resistant to chemotherapy or rapidly acquires this phenotype after chemotherapy exposure. In this study, we identified a docetaxel-induced resistance mechanism centered on CCL2. METHODS: We compared the gene expression profiles in individual human prostate cancer specimens before and after exposure to chemotherapy collected from previously untreated patients who participated in a clinical trial of preoperative chemotherapy. Subsequently, we used the gain- and loss-of-function approach in vitro to identify a potential mechanism underlying chemotherapy resistance. RESULTS: Among the molecular signatures associated with treatment, several genes that regulate the inflammatory response and chemokine activity were upregulated including a significant increase in transcripts encoding the CC chemokine CCL2. Docetaxel increased CCL2 expression in prostate cancer cell lines in vitro. CCL2-specific siRNA inhibited LNCaP and LAPC4 cell proliferation and enhanced the growth inhibitory effect of low-dose docetaxel. In contrast, overexpression of CCL2 or recombinant CCL2 protein stimulated prostate cancer cell proliferation and rescued cells from docetaxel-induced cytotoxicity. This protective effect of CCL2 was associated with activation of the ERK/MAP kinase and PI3K/AKT, inhibition of docetaxel-induced Bcl2 phosphorylation at serine 70, phosphorylation of Bad, and activation of caspase-3. The addition of a PI3K/AKT inhibitor Ly294002 reversed the CCL2 protection and was additive to docetaxel-induced toxicity. CONCLUSION: These results support a mechanism of chemotherapy resistance mediated by cellular stress responses involving the induction of CCL2 expression and suggest that inhibiting CCL2 activity could enhance therapeutic responses to taxane-based therapy.

CD90/THY1 is overexpressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker.

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared with non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, ∼5 to 10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma has a function in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells.

Prostate cancer-associated gene expression alterations determined from needle biopsies.

PURPOSE: To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. EXPERIMENTAL DESIGN: Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays composed of 6,200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative reverse transcription-PCR. RESULTS: Comparative analyses identified 954 transcript alterations associated with cancer (q < 0.01%), including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy use, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of androgen receptor expression changes was noted. In exploratory analyses, androgen receptor down-regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. CONCLUSIONS: Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.

Correlation between higher-order aberrations and visual acuity recovery (CoHORT) after spectacles treatment for pediatric refractive amblyopia: A pilot study using iDesign measurement.

PURPOSE: To determine the correlation between higher-order aberrations (HOAs) and best-corrected visual acuity (BCVA) recovery speed after spectacles treatment using iDesign measurements in refractive amblyopic children. METHODS: This is a prospective case series. Children aged from 3 to 7 years with refractive amblyopia (Landolt C equivalent < 0.8) were recruited. All participants were followed for at least 6 months after full correction of the refraction error by spectacles. The HOAs were measured using iDesign before and after cycloplegia at first visit and at 3-month intervals. Then correlation between BCVA recovery after treatment for 6 months and HOAs was determined. RESULTS: We analyzed 24 eyes of 12 children (mean age, 4.5 years). Baseline mean BCVA was logarithm of minimal angle of resolution (logMAR) 0.335 (Landolt C equivalent 0.46), which improved to logMAR 0.193 (Landolt C equivalent 0.64) after treatment with full-correction spectacles for 6 months. The amblyopic eye BCVA recovery was negatively correlated with tetrafoil with/without cycloplegia (P = 0.006 and 0.022, respectively) and trefoil with cycloplegia (P = 0.049). CONCLUSIONS: trefoil and tetrafoil measured with iDesign negatively correlates with the BCVA recovery speed of refractive amblyopic eyes after spectacles treatment in this pilot study. The current study results may aid in further investigation for diagnosis and treatment of refractory refractive and idiopathic amblyopia.

Assessment of the Feasibility and Safety of Durvalumab for Treatment of Solid Tumors in Patients With HIV-1 Infection: The Phase 2 DURVAST Study.

Importance: Therapies targeting the programmed cell death 1 (PD-1) receptor or its ligand (PD-L1), such as the humanized monoclonal antibody durvalumab, have shown durable clinical responses in several tumor types. However, concerns about the safety and feasibility of PD-1/PD-L1 blockade in HIV-1-infected individuals have led to the exclusion of these patients from clinical trials on cancer immunotherapies. Objective: To evaluate the feasibility and safety of durvalumab treatment in patients with advanced cancer and virologically controlled HIV-1 infection. Design, Setting, and Participants: The DURVAST study was a nonrandomized, open-label, phase 2 clinical trial in patients with any solid tumor type in which anti-PD-1 or anti-PD-L1 antibodies have approved indications or for which there are data of antitumoral activity with no other available curative therapy. All patients had basal undetectable plasma viremia while undergoing combination antiretroviral therapy. Interventions: Treatment consisted of intravenous infusion of durvalumab (1500 mg every 4 weeks) until disease progression or unacceptable toxic effects. Main Outcomes and Measures: Adverse events were graded with the use of the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Tumor response was evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1. Results: A total of 20 HIV-1-infected patients with advanced cancer were enrolled; 16 (80%) were male, the median (range) age was 54 (30-73) years, and 12 (60%) had progressed with previous cancer treatment lines. A median (range) of 4 (1-16) cycles of durvalumab were administered. Drug-related adverse events were observed in 50% of patients, and all were grade 1 and 2 (mainly diarrhea, asthenia, and arthromyalgia). Four of 16 response-evaluable patients (25%) had a partial response. Five patients (31%) had stable disease, including 4 with durable stable disease (disease control rate of 50%). CD4+ and CD8+ T-cell counts and plasma HIV-1 viremia remained stable throughout the study. Conclusions and Relevance: Durvalumab treatment was feasible and safe in HIV-1-infected patients with cancer receiving combination antiretroviral therapy. HIV-1-infected patients on suppressive antiretroviral therapy with advanced cancer should have access to cancer immunotherapy treatments. Trial Registration: ClinicalTrials.gov Identifier: NCT03094286.

Molecular alterations in prostate carcinomas that associate with in vivo exposure to chemotherapy: identification of a cytoprotective mechanism involving growth differentiation factor 15.

PURPOSE: To identify molecular alterations associating with in vivo exposure of prostate carcinoma to chemotherapy and assess functional roles modulating tumor response and resistance. EXPERIMENTAL DESIGN: Patients with high-risk localized prostate cancer (tumor-node-metastasis >or= T(2b) or prostate-specific antigen >or= 15 ng/mL or Gleason glade >or= 4+3) were enrolled into a phase II clinical trial of neoadjuvant chemotherapy with docetaxel and mitoxantrone followed by prostatectomy. Pretreatment prostate tissue was acquired by needle biopsy and posttreatment tissue was acquired by prostatectomy. Prostate epithelium was captured by microdissection, and transcript levels were quantitated by cDNA microarray hybridization. Gene expression changes associated with chemotherapy were determined by a random variance t test. Several were verified by quantitative reverse transcription PCR. In vitro analyses determining the influence of growth differentiation factor 15 (GDF15) on chemotherapy resistance were done. RESULTS: Gene expression changes after chemotherapy were measured in 31 patients who completed four cycles of neoadjuvant chemotherapy. After excluding genes shown previously to be influenced by the radical prostatectomy procedure, we identified 51 genes with significant transcript level alterations following chemotherapy. This group included several cytokines, including GDF15, chemokine (C-X-C motif) ligand 10, and interleukin receptor 1beta. Overexpression of GDF15 or exposure of prostate cancer cell lines to exogenous recombinant GDF15 conferred resistance to docetaxel and mitoxantrone. CONCLUSIONS: Consistent molecular alterations were identified in prostate cancer cells exposed to docetaxel and mitoxantrone chemotherapy. These alterations include transcripts encoding cytokines known to be regulated through the nuclear factor-kappaB pathway. Chemotherapy-induced cytokines and growth factors, such as GDF15, contribute to tumor cell therapy resistance and may serve as targets to improve responses.