Importance: Intravitreous bevacizumab (0.25 mg to 0.625 mg) is commonly used to treat type 1 retinopathy of prematurity (ROP), but there are concerns about systemic toxicity, particularly the risk of neurodevelopmental delay. A much lower dose may be effective for ROP while reducing systemic risk. Previously, after testing doses of 0.25 mg to 0.031 mg, doses as low as 0.031 mg were found to be effective in small cohorts of infants. Objective: To find the lowest dose of intravitreous bevacizumab effective for severe ROP. Design, Setting, and Participants: Between April 2017 and May 2019, 59 premature infants with type 1 ROP in 1 or both eyes were enrolled in a masked, multicenter, dose de-escalation study. In cohorts of 10 to 14 infants, 1 eye per infant received 0.016 mg, 0.008 mg, 0.004 mg, or 0.002 mg of intravitreous bevacizumab. Diluted bevacizumab was prepared by individual research pharmacies and delivered using 300-µL syringes with 5/16-inch, 30-guage fixed needles. Analysis began July 2019. Interventions: Bevacizumab intravitreous injections at 0.016 mg, 0.008 mg, 0.004 mg, or 0.002 mg. Main Outcomes and Measures: Success was defined as improvement by 4 days postinjection and no recurrence of type 1 ROP or severe neovascularization requiring additional treatment within 4 weeks. Results: Fifty-five of 59 enrolled infants had 4-week outcomes completed; the mean (SD) birth weight was 664 (258) g, and the mean (SD) gestational age was 24.8 (1.6) weeks. A successful 4-week outcome was achieved for 13 of 13 eyes (100%) receiving 0.016 mg, 9 of 9 eyes (100%) receiving 0.008 mg, 9 of 10 eyes (90%) receiving 0.004 mg, but only 17 of 23 eyes (74%) receiving 0.002 mg. Conclusions and Relevance: These data suggest that 0.004 mg may be the lowest dose of bevacizumab effective for ROP. Further investigation is warranted to confirm effectiveness of very low-dose intravitreous bevacizumab and its effect on plasma vascular endothelial growth factor levels and peripheral retinal vascularization.
Myelination facilitates rapid axonal conduction, enabling efficient communication across different parts of the nervous system. Here we examined mechanisms controlling myelination after injury and during axon regeneration in the central nervous system (CNS). Previously, we discovered multiple molecular pathways and strategies that could promote robust axon regrowth after optic nerve injury. However, regenerated axons remain unmyelinated, and the underlying mechanisms are elusive. In this study, we found that, in injured optic nerves, oligodendrocyte precursor cells (OPCs) undergo transient proliferation but fail to differentiate into mature myelination-competent oligodendrocytes, reminiscent of what is observed in human progressive multiple sclerosis. Mechanistically, we showed that OPC-intrinsic GPR17 signaling and sustained activation of microglia inhibit different stages of OPC differentiation. Importantly, co-manipulation of GPR17 and microglia led to extensive myelination of regenerated axons. The regulatory mechanisms of stage-dependent OPC differentiation uncovered here suggest a translatable strategy for efficient de novo myelination after CNS injury.
Recently, we discovered that the cosmetic preservatives, benzalkonium chloride and formaldehyde, are especially toxic to human meibomian gland epithelial cells (HMGECs). Exposure to these agents, at concentrations approved for human use, leads within hours to cellular atrophy and death. We hypothesize that these effects are not unique, and that other cosmetic preservatives also exert adverse effects on HMGECs. Such compounds include parabens, phenoxyethanol and chlorphenesin, which have been reported to be toxic to corneal and conjunctival epithelial cells, the liver and kidney, as well as to irritate the eye. To test our hypothesis, we examined the influence of parabens, phenoxyethanol and chlorphenesin on the morphology, signaling, survival, proliferation and lipid expression of immortalized (I) HMGECs. These cells were cultured under proliferating or differentiating conditions with varying concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin for up to 5 days. We monitored the signaling ability, appearance, number and neutral lipid content of the IHMGECs, as well as their lysosome accumulation. Our findings show that a 30-min exposure of IHMGECs to these preservatives results in a significant reduction in the activity of the Akt pathway. This effect is dose-dependent and occurs at concentrations equal to (chlorphenesin) and less than (all others) those dosages approved for human use. Further, a 24-h treatment of the IHMGECs with concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin close to, or at, the approved human dose induces cellular atrophy and death. At all concentrations tested, no preservative stimulated IHMGEC proliferation. Of particular interest, it was not possible to evaluate the influence of these preservatives, at close to human approved dosages, on IHMGEC differentiation, because the cells did not survive the treatment. In summary, our results support our hypothesis and show that methylparaben, ethylparaben, phenoxyethanol and chlorphenesin are toxic to IHMGECs.
Purpose: The identification of target pathways to block excessive angiogenesis while simultaneously restoring physiological vasculature is an unmet goal in the therapeutic management of ischemic retinopathies. pNaKtide, a cell-permeable peptide that we have designed by mapping the site of α1 Na/K-ATPase (NKA)/Src binding, blocks the formation of α1 NKA/Src/reactive oxygen species (ROS) amplification loops and restores physiological ROS signaling in a number of oxidative disease models. The aim of this study was to evaluate the importance of the NKA/Src/ROS amplification loop and the effect of pNaKtide in experimental ischemic retinopathy. Methods: Human retinal microvascular endothelial cells (HRMECs) and retinal pigment epithelium (ARPE-19) cells were used to evaluate the effect of pNaKtide on viability, proliferation, and angiogenesis. Retinal toxicity and distribution were assessed in those cells and in the mouse. Subsequently, the role and molecular mechanism of NKA/Src in ROS stress signaling were evaluated biochemically in the retinas of mice exposed to the well-established protocol of oxygen-induced retinopathy (OIR). Finally, pNaKtide efficacy was assessed in this model. Results: The results suggest a key role of α1 NKA in the regulation of ROS stress and the Nrf2 pathway in mouse OIR retinas. Inhibition of α1 NKA/Src by pNaKtide reduced pathologic ROS signaling and restored normal expression of hypoxia-inducible factor 1-α/vascular endothelial growth factor (VEGF). Unlike anti-VEGF agents, pNaKtide did promote retinal revascularization while inhibiting neovascularization and inflammation. Conclusions: Targeting α1 NKA represents a novel strategy to develop therapeutics that not only inhibit neovascularization but also promote physiological revascularization in ischemic eye diseases.
Retinal blood vessels provide the necessary energy, nutrients and oxygen in order to support visual function and remove harmful particles from blood, thus acting to protect neuronal cells. The homeostasis of the retinal vessels is important for the maintenance of retinal visual function. Neovascularization is the most common cause of blindness in patients with retinopathy. Previous studies have shown that inflammatory mediators are known key regulators in retinopathy, but their causal link has been elusive. Although inflammation is often thought to arise from inflammatory cells like macrophages, neutrophils, and resident microglia, retinal neurons have also been reported to contribute to inflammation, through inflammatory signals, which mediate blood vessel growth. Therefore, it is important to explore the detailed mechanisms of neuroinflammation's effects on retinal neovascularization. This review looks to summarize current research on the relationship between retinal angiogenesis and neuroinflammation in retinopathy, as well as the potential effects of neuroinflammation on retinal neovascularization in different animal models.
PURPOSE: Lubricin, a boundary lubricant, is the body's unique antiadhesive, antifibrotic, antifriction, and antiinflammatory glycoprotein. This amphiphile is produced by numerous tissues and acts to regulate a number of processes, such as homeostasis, shear stress, tissue development, innate immunity, inflammation, and wound healing. We hypothesize that lubricin is also synthesized and expressed by the amniotic membrane (AM), which also possesses antiadhesive, antifibrotic, and antiinflammatory properties. We also hypothesize that lubricin, at least in part, mediates these AM capabilities. Our goal was to test our hypothesis. METHODS: We obtained multiple samples of fresh, cryopreserved (CP), and freeze-dried (FD) human AMs, as well as fresh placental tissue as positive controls, and processed them for light microscopy, immunofluorescence, and western blot analyses. We also evaluated the ability of recombinant human lubricin to associate with FD-AMs. RESULTS: Our results demonstrate that all fresh placental, fresh AM, and CP-AM samples contained lubricin. Lubricin was expressed in placental chorionic villi, AM epithelial and stromal cells, and CP-AM epithelia. No lubricin could be detected in FD-AMs but could be restored in FD-AMs after overnight incubation with recombinant human lubricin. CONCLUSIONS: This study supports our hypothesis that lubricin is expressed in human AMs. In addition, our data show that preservation methods influence the extent of this expression. Indeed, the disappearance of lubricin in FD-AMs may explain why dried AM reportedly loses its antiinflammatory and antiscarring abilities. It is possible that lubricin may mediate, at least in part, many of the biological properties of AMs.
Retinitis pigmentosa (RP) is a genetically heterogenous group of eye diseases in which initial degeneration of rods triggers secondary degeneration of cones, leading to significant loss of daylight, color, and high-acuity vision. Gene complementation with adeno-associated viral (AAV) vectors is one strategy to treat RP. Its implementation faces substantial challenges, however; for example, the tremendous number of loci with causal mutations. Gene therapy targeting secondary cone degeneration is an alternative approach that could provide a much-needed generic treatment for many patients with RP. Here, we show that microglia are required for the upregulation of potentially neurotoxic inflammatory factors during cone degeneration in RP, creating conditions that might contribute to cone dysfunction and death. To ameliorate the effects of such factors, we used AAV vectors to express isoforms of the antiinflammatory cytokine transforming growth factor beta (TGF-β). AAV-mediated delivery of TGF-β1 rescued degenerating cones in 3 mouse models of RP carrying different pathogenic mutations. Treatment with TGF-β1 protected vision, as measured by 2 behavioral assays, and could be pharmacologically disrupted by either depleting microglia or blocking the TGF-β receptors. Our results suggest that TGF-β1 may be broadly beneficial for patients with cone degeneration, and potentially other forms of neurodegeneration, through a pathway dependent upon microglia.
Importance: Although the central visual field (VF) in end-stage glaucoma may substantially vary among patients, structure-function studies and quality-of-life assessments are impeded by the lack of appropriate characterization of end-stage VF loss. Objective: To provide a quantitative characterization and classification of central VF loss in end-stage glaucoma. Design, Setting, and Participants: This retrospective cohort study collected data from 5 US glaucoma services from June 1, 1999, through October 1, 2014. A total of 2912 reliable 10-2 VFs of 1103 eyes from 1010 patients measured after end-stage 24-2 VFs with a mean deviation (MD) of -22 dB or less were included in the analysis. Data were analyzed from March 28, 2018, through May 23, 2019. Main Outcomes and Measures: Central VF patterns were determined by an artificial intelligence algorithm termed archetypal analysis. Longitudinal analyses were performed to investigate whether the development of central VF defect mostly affects specific vulnerability zones. Results: Among the 1103 patients with the most recent VFs, mean (SD) age was 70.4 (14.3) years; mean (SD) 10-2 MD, -21.5 (5.6) dB. Fourteen central VF patterns were determined, including the most common temporal sparing patterns (304 [27.5%]), followed by mostly nasal loss (280 [25.4%]), hemifield loss (169 [15.3%]), central island (120 [10.9%]), total loss (91 [8.3%]), nearly intact field (56 [5.1%]), inferonasal quadrant sparing (42 [3.8%]), and nearly total loss (41 [3.7%]). Location-specific median total deviation analyses partitioned the central VF into a more vulnerable superonasal zone and a less vulnerable inferotemporal zone. At 1-year and 2-year follow-up, new defects mostly occurred in the more vulnerable zone. Initial encroachments on an intact central VF at follow-up were more likely to be from nasal loss (11 [18.4%]; P < .001). One of the nasal loss patterns had a substantial chance at 2-year follow-up (8 [11.0%]; P = .004) to shift to total loss, whereas others did not. Conclusions and Relevance: In this study, central VF loss in end-stage glaucoma was found to exhibit characteristic patterns that might be associated with different subtypes. Initial central VF loss is likely to be nasal loss, and 1 specific type of nasal loss is likely to develop into total loss.
OBJECTIVE: The diagnosis of sarcoid optic neuropathy is time-sensitive, as delayed treatment risks irreversible vision loss. We sought to analyze its characteristics and outcomes. METHODS: We performed a multi-center retrospective study of sarcoid optic neuropathy among 5 USA medical centers. Inclusion criteria were: 1) clinical optic neuropathy; 2) optic nerve/sheath enhancement on neuroimaging; 3) pathological confirmation of systemic or nervous system sarcoidosis. RESULTS: Fifty-one patients were included. The median onset age of sarcoid optic neuropathy was 50 years (range, 17-70 years) and 71% were female. The median visual acuity at nadir in the most affected eye was 20/80 (range, 20/20 to no-light-perception). Thirty-four of 50 (68%) patients had radiologic evidence of other nervous system involvement and 20 (39%) patients had symptoms/signs of other cranial nerve dysfunction. Cerebrospinal fluid analysis revealed an elevated white blood cell count in 22 of 31 (71%) patients (median: 14/μL; range: 1-643/μL). Pathologic confirmation of sarcoidosis was by biopsy of systemic/pulmonary site, 34 (67%); optic nerve/sheath, 9 (18%); or other nervous system region, 8 (16%). Forty patients improved with treatment (78%), 98% receiving corticosteroids and 65% receiving steroid-sparing immunosuppressants, yet 11/46 patients (24%) had a visual acuity of 20/200 or worse at last follow-up. CONCLUSIONS: Sarcoid optic neuropathy frequently occurs with other clinical and radiologic abnormalities caused by neurosarcoidosis and diagnostic confirmation occasionally requires optic nerve/sheath biopsy. Improvement with treatment is common but most patients have some residual visual disability. Improved recognition and a more expeditious diagnosis and treatment may spare patients from permanent vision loss.
Whitman MC, Di Gioia SA, Chan W-M, Gelber A, Pratt BM, Bell JL, Collins TE, Knowles JA, Armoskus C, Pato M, Pato C, Shaaban S, Staffieri S, MacKinnon S, Maconachie GDE, Elder JE, Traboulsi EI, Gottlob I, Mackey DA, Hunter DG, Engle EC, Engle EC. Recurrent Rare Copy Number Variants Increase Risk for Esotropia. Invest Ophthalmol Vis Sci 2020;61(10):22.Abstract
Purpose: To determine whether rare copy number variants (CNVs) increase risk for comitant esotropia. Methods: CNVs were identified in 1614 Caucasian individuals with comitant esotropia and 3922 Caucasian controls from Illumina SNP genotyping using two Hidden Markov model (HMM) algorithms, PennCNV and QuantiSNP, which call CNVs based on logR ratio and B allele frequency. Deletions and duplications greater than 10 kb were included. Common CNVs were excluded. Association testing was performed with 1 million permutations in PLINK. Significant CNVs were confirmed with digital droplet polymerase chain reaction (ddPCR). Whole genome sequencing was performed to determine insertion location and breakpoints. Results: Esotropia patients have similar rates and proportions of CNVs compared with controls but greater total length and average size of both deletions and duplications. Three recurrent rare duplications significantly (P = 1 × 10-6) increase the risk of esotropia: chromosome 2p11.2 (hg19, 2:87428677-87965359), spanning one long noncoding RNA (lncRNA) and two microRNAs (OR 14.16; 95% confidence interval [CI] 5.4-38.1); chromosome 4p15.2 (hg19, 4:25554332-25577184), spanning one lncRNA (OR 11.1; 95% CI 4.6-25.2); chromosome 10q11.22 (hg19, 10:47049547-47703870) spanning seven protein-coding genes, one lncRNA, and four pseudogenes (OR 8.96; 95% CI 5.4-14.9). Overall, 114 cases (7%) and only 28 controls (0.7%) had one of the three rare duplications. No case nor control had more than one of these three duplications. Conclusions: Rare CNVs are a source of genetic variation that contribute to the genetic risk for comitant esotropia, which is likely polygenic. Future research into the functional consequences of these recurrent duplications may shed light on the pathophysiology of esotropia.
We tested younger and older observers' attention and long-term memory functions in a "hybrid search" task, in which observers look through visual displays for instances of any of several types of targets held in memory. Apart from a general slowing, search efficiency did not change with age. In both age groups, reaction times increased linearly with the visual set size and logarithmically with the memory set size, with similar relative costs of increasing load (Experiment 1). We replicated the finding and further showed that performance remained comparable between age groups when familiarity cues were made irrelevant (Experiment 2) and target-context associations were to be retrieved (Experiment 3). Our findings are at variance with theories of cognitive aging that propose age-specific deficits in attention and memory. As hybrid search resembles many real-world searches, our results might be relevant to improve the ecological validity of assessing age-related cognitive decline.
The damage or loss of retinal ganglion cells (RGCs) and their axons accounts for the visual functional defects observed after traumatic injury, in degenerative diseases such as glaucoma, or in compressive optic neuropathies such as from optic glioma. By using optic nerve crush injury models, recent studies have revealed the cellular and molecular logic behind the regenerative failure of injured RGC axons in adult mammals and suggested several strategies with translational potential. This review summarizes these findings and discusses challenges for developing clinically applicable neural repair strategies.
PURPOSE: The purpose of this assessment is to evaluate the accuracy of autorefraction compared with cycloplegic retinoscopy in children. METHODS: Literature searches were last conducted in October 2019 in the PubMed and the Cochrane Library databases for studies published in English. The combined searches yielded 118 citations, of which 53 were reviewed in full text. Of these, 31 articles were deemed appropriate for inclusion in this assessment and subsequently assigned a level of evidence rating by the panel methodologists. Four articles were rated level I, 11 were rated level II, and 16 were rated level III articles. The 16 level III articles were excluded from this review. RESULTS: Thirteen of the 15 studies comparing cycloplegic autorefraction with cycloplegic retinoscopy found a mean difference in spherical equivalent or sphere of less than 0.5 diopters (D); most were less than 0.25 D. Even lower mean differences were found when evaluating the cylindrical component of cycloplegic autorefraction versus cycloplegic retinoscopy. Despite low mean variability, there was significant individual measurement variability; the 95% limits of agreement were wide and included clinically relevant differences. Comparisons of noncycloplegic with cycloplegic autorefractions found that noncyloplegic refraction tends to over minus by 1 to 2 D. CONCLUSIONS: Cycloplegic autorefraction is appropriate to use in pediatric population-based studies. Cycloplegic retinoscopy can be valuable in individual clinical cases to confirm the accuracy of cycloplegic autorefraction, particularly when corrected visual acuity is worse than expected or the autorefraction results are not consistent with expected findings.
BACKGROUND: Advanced age-related macular degeneration (AMD) is a leading cause of blindness. While around half of the genetic contribution to advanced AMD has been uncovered, little is known about the genetic architecture of early AMD. METHODS: To identify genetic factors for early AMD, we conducted a genome-wide association study (GWAS) meta-analysis (14,034 cases, 91,214 controls, 11 sources of data including the International AMD Genomics Consortium, IAMDGC, and UK Biobank, UKBB). We ascertained early AMD via color fundus photographs by manual grading for 10 sources and via an automated machine learning approach for > 170,000 photographs from UKBB. We searched for early AMD loci via GWAS and via a candidate approach based on 14 previously suggested early AMD variants. RESULTS: Altogether, we identified 10 independent loci with statistical significance for early AMD: (i) 8 from our GWAS with genome-wide significance (P < 5 × 10), (ii) one previously suggested locus with experiment-wise significance (P < 0.05/14) in our non-overlapping data and with genome-wide significance when combining the reported and our non-overlapping data (together 17,539 cases, 105,395 controls), and (iii) one further previously suggested locus with experiment-wise significance in our non-overlapping data. Of these 10 identified loci, 8 were novel and 2 known for early AMD. Most of the 10 loci overlapped with known advanced AMD loci (near ARMS2/HTRA1, CFH, C2, C3, CETP, TNFRSF10A, VEGFA, APOE), except two that have not yet been identified with statistical significance for any AMD. Among the 17 genes within these two loci, in-silico functional annotation suggested CD46 and TYR as the most likely responsible genes. Presence or absence of an early AMD effect distinguished the known pathways of advanced AMD genetics (complement/lipid pathways versus extracellular matrix metabolism). CONCLUSIONS: Our GWAS on early AMD identified novel loci, highlighted shared and distinct genetics between early and advanced AMD and provides insights into AMD etiology. Our data provide a resource comparable in size to the existing IAMDGC data on advanced AMD genetics enabling a joint view. The biological relevance of this joint view is underscored by the ability of early AMD effects to differentiate the major pathways for advanced AMD.
PURPOSE: To review the literature on the efficacy of intense pulsed light (IPL) on the eyelids in the management of meibomian gland disease (MGD) and meibomian gland-related ocular surface disease. METHODS: A literature search was last conducted on May 15, 2019, in the PubMed and Cochrane Library databases for English-language original research that assessed the effect of IPL on MGD in adult patients. Thirty-three articles were identified, and 12 studies were determined to be relevant to the criteria outlined for assessment. The panel methodologist (V.K.A.) assigned a level of evidence rating to each study; 4 studies were rated level II, and 8 studies were rated level III. Five studies had potential conflicts of interest and design limitations that affected interpretation of results. RESULTS: All studies documented improvement in clinically meaningful metrics, including tear breakup time (TBUT), corneal staining and eyelid margin measurements, meibum quality, meibomian gland expressability, ocular surface disease index (OSDI), and standard patient evaluation of eye dryness (SPEED) questionnaire scores. Side effects were relatively uncommon but included discomfort, cutaneous erythema, blistering, eyelash loss, and floaters; these were uniformly self-limited. CONCLUSIONS: Although methodological limitations and potential conflicts of interest in some studies raised concern, the existing body of literature demonstrates improvements in the signs and symptoms of MGD after IPL therapy.
In visual search tasks, observers look for targets among distractors. In the lab, this often takes the form of multiple searches for a simple shape that may or may not be present among other items scattered at random on a computer screen (e.g., Find a red T among other letters that are either black or red.). In the real world, observers may search for multiple classes of target in complex scenes that occur only once (e.g., As I emerge from the subway, can I find lunch, my friend, and a street sign in the scene before me?). This article reviews work on how search is guided intelligently. I ask how serial and parallel processes collaborate in visual search, describe the distinction between search templates in working memory and target templates in long-term memory, and consider how searches are terminated.
BACKGROUND/PURPOSE: To report a unique case of a pulmonary carcinoid tumor unilaterally metastatic to the iris and ciliary body and bilaterally to the choroid that was conservatively followed. METHODS: A 46-year-old woman presented with bilateral choroidal lesions and a left iris tumor. Ultrasound biomicroscopy disclosed a ciliary body component. A diagnosis of metastatic carcinoid tumor was made based on the clinical features. Rather than an excision, photodynamic therapy, or radiation treatment, as has been reported in all previous cases of carcinoid tumor metastatic to the iris, the patient was observed. RESULTS: Excellent vision was maintained for 8 years. The iris tumor gradually enlarged, but the choroidal lesions remained unchanged. The iris with the carcinoid tumor gradually acquired a brown pigmentation; this is the first reported case of acquired iris heterochromia in the setting of carcinoid tumor. CONCLUSION: We conclude, in cases of metastatic carcinoid in which visual acuity is excellent and the patient is asymptomatic, that observation of the ocular lesions is an acceptable course of action. The iris heterochromia is believed to have been caused by secretory factors produced by the tumor.
PURPOSE: To determine if there is a biologic rationale for using checkpoint inhibitor drugs targeting programmed cell death ligand 1 (PD-L1) and PD-L2 in the treatment of adenoid cystic carcinoma of the orbit. METHODS: Twenty-three cases of adenoid cystic carcinoma involving the orbit (13 primary lacrimal gland, 5 secondarily extending into the orbit, and 5 unspecified) were examined histopathologically. Immunohistochemistry for PD-L1, PD-L2, and CD8 was performed. Charts were reviewed for clinical correlations. RESULTS: Expression of PD-L1 and of PD-L2 was overall low in adenoid cystic carcinoma (mean expression 1.4 ± 0.9 of 5 for PD-L1, mean 0.83 ± 1.1 of 5 for PD-L2), and tumor-infiltrating CD8-positive T-lymphocytes were sparse (mean 1.1 ± 0.51 of 3). Only 13 of the 23 (57%) cases expressed PD-L1 as a combined positive score ≥1 of cells. No associations were found between expression levels of these markers and patient sex, tumor site of origin, Tumor, Node, Metastasis stage, or patient outcome. A significant association was observed between stromal PD-L1 expression and tumor histopathologic subtype (p = 0.05), and between tumor PD-L1 expression and prior exposure to radiation (p = 0.03). CONCLUSIONS: Checkpoint inhibitor drugs may have limited impact in the treatment and clinical course of orbital adenoid cystic carcinoma based on the low frequency of CD8 infiltrate and low expression of PD-L1 and PD-L2. Pretreatment with radiation, however, may improve tumor response to checkpoint inhibitor drugs.