Gain of function of ASXL1 truncating protein in the pathogenesis of myeloid malignancies.

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

Expression and regulation of alarmin cytokine IL-1α in human retinal pigment epithelial cells.

Human retinal pigment epithelial (hRPE) cells play important immune-regulatory roles in a variety of retinal pathologic processes, including the production of inflammatory cytokines that are essential mediators of the innate immune response within the ocular microenvironment. The pro-inflammatory "alarmin" cytokine IL-1α has been implicated in both infectious and non-infectious retinal diseases, but its regulation in the retina is poorly understood. The purpose of this study was to elucidate the expression and regulation of IL-1α within hRPE cells. To do this, IL-1α mRNA and protein in hRPE cells was assessed by RT-PCR, qPCR, ELISA, Western blot, and immunofluorescence following treatment with a variety of stimuli and inhibitors. ER stress, LPS, IL-1ß, and TLR2 activation all significantly increased intracellular IL-1α protein. Increasing intracellular calcium synergized both LPS- and Pam3CSK4-induced IL-1α protein production. Accordingly, blocking calcium signaling and calpain activity strongly suppressed IL-1α protein expression. Significant but more moderate inhibition occurred following blockage of TLR4, caspase-4, or caspase-1. Neutralizing antibodies to IL-1ß and TLR2 partially eliminated LPS- and TLR2 ligand Pam3CSK4-stimulated IL-1α protein production. IFN-ß induced caspase-4 expression and activation, and also potentiated LPS-induced IL-1α expression, but IFN-ß alone had no effect on IL-1α protein production. Interestingly, all inhibitors targeting the PI3K/Akt pathway, with the exception of Ly294002, strongly increased IL-1α protein expression. This study improves understanding of the complex mechanisms regulating IL-1α protein expression in hRPE cells by demonstrating that TLR4 and TLR2 stimulation and exposure to IL-1ß, ER stress and intracellular calcium all induce hRPE cells to produce intracellular IL-1α, which is negatively regulated by the PI3K/Akt pathway. Additionally, the non-canonical inflammasome pathway was shown to be involved in LPS-induced hRPE IL-1α expression through caspase-4 signaling.

Distinct CD40L receptors mediate inflammasome activation and secretion of IL-1ß and MCP-1 in cultured human retinal pigment epithelial cells.

CD40L signaling occurs in several diseases with inflammatory components, including ocular and retinal diseases. However, it has never been evaluated as a pathogenic mechanism in age-related macular degeneration (AMD) or as an inducer of inflammasome formation in any cell type. mRNA and protein levels of CD40, IL-1ß, NALP1, NALP3, caspase-1, and caspase-5 were determined by RT-PCR, qPCR, and Western blot. CD40L receptor (CD40, α5ß1, and CD11b) expression was determined by Western and immunofluorescent staining. IL-1ß, IL-18, and MCP-1 secretions were determined by ELISA. NALP1 and NALP3 inflammasome formation were determined by Co-IP. Experiments were conducted on primary human retinal pigment epithelial (hRPE) cells from four different donors. Human umbilical vein endothelial (HUVEC) and monocytic leukemia (THP-1) cells demonstrated the general applicability of our findings. In hRPE cells, CD40L-induced NALP1 and NALP3 inflammasome activation, cleavage of caspase-1 and caspase-5, and IL-1ß and IL-18 secretion. Interestingly, neutralizing CD11b and α5ß1 antibodies, but not CD40, reduced CD40L-induced IL-1ß secretion in hRPE cells. Similarly, CD40L treatment also induced HUVEC and THP-1 cells to secret IL-1ß through CD11b and α5ß1. Additionally, the CD40L-induced IL-1ß secretion acted in an autocrine/paracrine manner to feed back and induce hRPE cells to secrete MCP-1. This study is the first to show that CD40L induces inflammasome activation in any cell type, including hRPE cells, and that this induction is through CD11b and α5ß1 cell-surface receptors. These mechanisms likely play an important role in many retinal and non-retinal diseases and provide compelling drug targets that may help reduce pro-inflammatory processes.

Recent developments in prognostic and predictive testing in uveal melanoma.

PURPOSE OF REVIEW: To provide an update on the rapidly evolving methods for assessing prognosis and predicting response to targeted molecular therapy in uveal melanoma. RECENT FINDINGS: The techniques for assessing prognosis in uveal melanoma have evolved from simple physical features, such as tumor size, location, and cell morphology, to the slightly more sophisticated counting of chromosomal gains and losses. More recently, gene expression profiling has provided a highly accurate and biologically informative gold standard for molecular prognostication. The latest step in the evolution of molecular testing has been the recent discovery of major driver mutations that allow predictive testing of response to targeted molecular therapies. Mutations in GNAQ and GNA11 are early events that promote cell proliferation, and these mutations are sensitive to MAPK kinase, PKC, and AKT inhibitors. Mutations in BAP1, SF3B1, and EIF1AX are later events that are largely mutually exclusive. Mutations in BAP1 are strongly associated with metastasis, whereas those in SF3B1 and EIF1AX are associated with good prognosis. Uveal melanomas with BAP1 mutations demonstrate sensitivity to epigenetic modulators, such as histone deacetylase inhibitors. Clinical trials are now available to evaluate the efficacy of these targeted molecular agents in patients with uveal melanoma. SUMMARY: Molecular prognostic testing and enrollment of high-risk patients into clinical trials of targeted molecular therapy are rapidly becoming the standard of care in the management of uveal melanoma.

Traumatic cataract induced by improper use of a percussion massage gun.

Purpose: We describe a case of traumatic cataract after improper use of a percussion massage gun over the periorbital area. Observations: A 38-year-old female with a history of high myopia and fibromyalgia presented to the emergency department with painless monocular vision loss OS, noticed two days prior and described as a "white film" over her eye. BCVA was 20/20 OD and 20/600 OS. IOP was normal. Slit lamp examination OS showed a dense posterior subcapsular cataract in a rosette pattern without signs of zonular instability. B-scan ultrasonography showed a clear vitreous cavity without structural globe anomalies. No other abnormalities were apparent. After ruling out other causes, vision loss was attributed to development of a traumatic cataract secondary to percussive massage gun use over the left temple and periorbital area, including directly over the eye, during the past few weeks as an attempt to relieve intractable headaches. Conclusion and importance: Improper use of massage guns can lead to severe ocular side effects including traumatic cataracts that may be difficult to manage. There is a need to educate patients about potential harms as well as require manufacturers to clearly display safety information.

Retrospective Optimization of the Hawkeye Orbital Fracture Prioritization and Evaluation Algorithms for Triaging Ophthalmic Care.

Objectives: Many orbital fracture patients are transferred to tertiary care centers for immediate ophthalmology consultation, though few require urgent ophthalmic evaluation or intervention. This overutilizes limited resources and overburdens patients and the health care system with travel and emergency department (ED) expenses. A simple, easy-to-use, clinical decision-making tool is needed to aid local EDs and triage services in effectively identifying orbital fracture patients who need urgent ophthalmic evaluation. Design: Single center, retrospective cohort study. Subjects: Orbital fracture patients aged ≥ 18 years who presented to the study institution's emergency department and received an ophthalmology consultation. Methods: Ocular injuries that required close monitoring or an intervention within the first few hours after presentation were termed urgent. Two Hawkeye Orbital Fracture Prioritization and Evaluation (HOPE) algorithms were developed to identify orbital fracture patients needing urgent evaluation; including 1 algorithm incorporating computerized tomography (CT) scans interpreted by ophthalmology (HOPE+CT). Algorithms were compared with 3 previously published protocols: the University of Texas Health Science Center at Houston (UTH), the South Texas Orbital Fracture Protocol (STOP), and Massachusetts Eye and Ear (MEE) algorithms. Main Outcome Measures: Correct triage of patients with orbital fractures who have urgent ocular or orbital conditions. Results: In the study institution's ED, 134 adult patients (145 orbits) were seen with orbital fractures in 2019. Eighteen (13.4%) had ocular or orbital conditions categorized as urgent. The HOPE tool resulted in 100% sensitivity and 78.4% specificity. The HOPE+CT tool resulted in 100.0% sensitivity and 94.0% specificity. The UTH algorithm was 91.7% sensitive and 76.5% specific. South Texas Orbital Fracture Protocol and MEE were both 100% sensitive but only 35.1% and 32.8% specific, respectively. Conclusions: The HOPE and HOPE+CT algorithms were superior or equal to the UTH, STOP, and MEE algorithms in terms of specificity while detecting all urgent cases. Implementation of a triage protocol that uses the HOPE or HOPE+CT algorithms could improve resource utilization and reduce health care costs through identification of orbital fracture patients needing urgent evaluation. An online tool that deploys the HOPE+CT algorithm in a user-friendly interface has been developed and is undergoing prospective validation before public dissemination. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME than can be targeted therapeutically in cancer.

Successful response to first-line treatment with osimertinib for choroidal metastasis from EGFR-mutated non-small-cell lung cancer.

Purpose: Describe the use of osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, as the first-line treatment in a patient with choroidal and central nervous system metastases from EGFR-mutated non-small cell lung cancer. Observations: A 68-year-old man presented with an amelanotic choroidal lesion in the left eye concerning for choroidal metastasis. Systemic evaluation identified widely metastatic adenocarcinoma of the lung with EGFR exon 19 mutation. Within one month of initiating treatment with osimertinib, there was complete resolution of the subretinal fluid over the choroidal lesion and decreased thickness of the lesion. At follow-up after three months of treatment, the lesion was clinically involuted. Positron emission tomography at two months and magnetic resonance imaging of the brain at three months showed significant interval decrease in size and activity of the primary right lung lesion, central nervous system lesions, and other metastatic sites with no new metastatic lesions. After 17 months of follow up, the lesion remained involuted. Conclusions and Importance: Osimertinib may be considered as a first-line treatment option in patients with choroidal metastases from an EGFR-mutated non-small cell lung cancer.

RB1 loss triggers dependence on ESRRG in retinoblastoma.

Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 20 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify previously unknown Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death, which is exacerbated in hypoxia. These findings reveal a previously unidentified dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.

Retinal flavoprotein fluorescence correlates with mitochondrial stress, apoptosis, and chemokine expression.

Oxidative stress and mitochondrial dysfunction occur before apoptosis in many retinal diseases. Under these conditions, a larger fraction of flavoproteins become oxidized and, when excited by blue-light, emit green flavoprotein fluorescence (FPF). In this study, we evaluated the utility of FPF as an early indicator of mitochondrial stress, pre-apoptotic cellular instability, and apoptosis of human retinal pigment epithelial (HRPE) cells subjected to hydrogen peroxide (H(2)O(2)) or monocytes (unstimulated or interferon-γ-stimulated) in vitro and of freshly-isolated pieces of human and rat neural retina subjected to H(2)O(2)ex vivo. Increased FPF of HRPE cells exposed to H(2)O(2) correlated with reduced mitochondrial membrane potential (ΔΨm) and increased apoptosis in a time- and dose-dependent manner. HRPE cells co-cultured with monocytes had increased FPF that correlated in a time-dependent manner with reduced ΔΨm, increased apoptosis, and early expression of pro-inflammatory chemokines, interleukin-8 (IL8) and monocyte chemotactic factor-1 (MCP1), which are known to be induced by oxidative stress. Increased FPF, reduced ΔΨm, and upregulation of IL8 and MCP1 occurred as early as 1-2 h after exposure to stressors, while apoptosis did not occur in HRPE cells until later time points. The antioxidant, N-acetyl-cysteine (NAC), inhibited increased FPF and apoptosis of HRPE cells subjected to H(2)O(2). Increased FPF of human and rat neural retina also correlated with increased apoptosis. This study suggests that FPF is a useful measure of mitochondrial function in retinal cells and tissues and can detect early mitochondrial dysfunction that may precede apoptosis.

Impact of reduced elective ophthalmic surgical volume on U.S. hospitals during the early coronavirus disease 2019 pandemic.

PURPOSE: To estimate the financial impact of coronavirus disease 2019 (COVID-19)-related shutdowns on ophthalmic surgery performed at hospital outpatient departments (HOPDs) in the United States. SETTING: Nationally representative sample of U.S. hospital payment and cost data. DESIGN: Retrospective review and economic impact analysis. METHODS: The Nationwide Ambulatory Surgery Sample (NASS) was used to identify ophthalmic surgical procedures and associated charges, which were performed at HOPDs. The highest volume elective ophthalmic procedures were identified. The total hospital cost and payment amount was calculated for each procedure using the Hospital Outpatient Prospective Payment System (OPPS) maintained by the Centers for Medicare & Medicaid Services. Net facility income (estimated payments less OPPS rates) was determined for each elective surgical procedure category and stratified by hospital teaching status. RESULTS: In 2017, elective cataract, strabismus, and keratoplasty surgeries were performed 1 230 992 times at HOPDs. The total cost of these elective surgeries was 2350 million U.S. dollars (USD), with a total hospital payment of 3624 to 3786 million USD. This led to an estimated net income of 1278 to 1440 million USD overall to U.S. hospitals in the NASS dataset from elective ophthalmic surgery (approximately 107 to 120 million USD per month), with a larger proportion performed in teaching hospitals. CONCLUSIONS: The cessation of elective ophthalmic surgeries at HOPDs during COVID-19 resulted in a significant loss of income for hospitals in the United States and teaching experiences for trainees at academic medical centers.

Whole exome profiling and mutational analysis of Ocular Surface Squamous Neoplasia.

PURPOSE: To determine genetic mutational profiles in patients with Ocular Surface Squamous Neoplasia (OSSN) using whole exome sequencing. METHODS: Prospective, case-series study. Patient recruitment was conducted in a single tertiary referral center from April to September 2017. Specimens were obtained by incisional biopsies of tumors from ten eyes with histopathologic confirmation of OSSN. DNA whole exome sequencing and mutation analysis were performed. RESULTS: Ten patients with clinically-diagnosed OSSN underwent DNA whole exome sequencing analysis. Deleterious mutations in 305 genes known to drive tumor development and progression were found. These mutations centered around two main pathways: DNA repair/cell cycle and development/growth. All ten samples had at least one mutation in a DNA repair/cell cycle gene and all but one sample had one in a development/growth gene. The most common mutation was found in TP53 and HGF (both present in 50% of cases) and mutually exclusive mutations were found in BRCA1 and BRCA2 (50% of cases). Mutations in APC, MSH6, PDGFRA, and PTCH1 were found in 40% of cases. Global mutation analysis identified ultraviolet induced radiation as the only mutational signature present in the dataset. CONCLUSIONS: Mutations found in samples from patients with OSSN are mainly induced by ultraviolet radiation and occur within two main pathways related to DNA repair/cell cycle and development/growth. There are many clinically available drugs and several others being evaluated in clinical trials that target the genes found mutated in this study, offering new therapeutic options for OSSN.

Detection of retinal metabolic stress resulting from central serous retinopathy.

PURPOSE: To test whether eyes with central serous retinopathy have elevated retinal flavoprotein fluorescence (FPF) using a novel clinical imaging method. METHODS: Three male patients with unilateral central serous retinopathy were examined for FPF at 535 nm induced by 1-msec flashes of 467 nm light. FPF was captured with an electron multiplying charged-coupled device camera with a 512 x 512 pixel chip. Average intensity of retinal FPF for each affected eye was compared with the contralateral, unaffected eye and with six age-matched control eyes by analyzing histograms of pixel intensities plotted for each eye. RESULTS: For each patient, the central serous retinopathy-affected eye had a significantly greater retinal FPF when compared with the retinal FPF of the unaffected eye. Eyes affected with central serous retinopathy had retinal FPF values that averaged 98% greater than the retinal FPF of age-matched control eyes. CONCLUSION: Flavoprotein fluorescence analysis may be useful for rapidly and noninvasively identifying metabolic tissue stress of central serous retinopathy.

BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome. It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression. Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development. Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma. The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4. Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion. These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.

BAP1 Loss Is Associated with DNA Methylomic Repatterning in Highly Aggressive Class 2 Uveal Melanomas.

PURPOSE: The strong association between BAP1 mutations and metastasizing Class 2 uveal melanoma (UM) suggests that epigenetic alterations may play a significant role in tumor progression. Thus, we characterized the impact of BAP1 loss on the DNA methylome in UM.Experimental Design: Global DNA methylation was analyzed in 47 Class 1 and 45 Class 2 primary UMs and in UM cells engineered to inducibly deplete BAP1. RNA-Seq was analyzed in 80 UM samples and engineered UM cells. RESULTS: Hypermethylation on chromosome 3 correlated with downregulated gene expression at several loci, including 3p21, where BAP1 is located. Gene set analysis of hypermethylated and downregulated genes identified axon guidance and melanogenesis as deregulated pathways, with several of these genes located on chromosome 3. A novel hypermethylated site within the BAP1 locus was found in all Class 2 tumors, suggesting that BAP1 itself is epigenetically regulated. Highly differentially methylated probes were orthogonally validated using bisulfite sequencing, and they successfully distinguished Class 1 and Class 2 tumors in 100% of cases. In functional validation experiments, BAP1 knockdown in UM cells induced methylomic repatterning similar to UM tumors, enriched for genes involved in axon guidance, melanogenesis, and development. CONCLUSIONS: This study, coupled with previous work, suggests that the initial event in the divergence of Class 2 UM from Class 1 UM is loss of one copy of chromosome 3, followed by mutation of BAP1 on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs.

Genomic evolution of uveal melanoma arising in ocular melanocytosis.

Ocular melanocytosis is the most important predisposing condition for the eye cancer uveal melanoma (UM). Here, we present a patient who developed UM arising within ocular melanocytosis who was treated with enucleation (eye removal), which provided an invaluable opportunity to interrogate both the UM and adjacent uveal tissue containing the melanocytosis using whole-exome and deep-targeted sequencing. This analysis revealed a clonal PLCB4 mutation in the melanocytosis, confirming that this is indeed a neoplastic condition and explaining why it predisposes to UM. This mutation was present in 100% of analyzed UM cells, indicating that a PLCB4-mutant cell gave rise to the UM. The earliest aberrations specific to the tumor were loss of Chromosomes 1p, 3, and 9p, which were present in virtually all tumor cells. A mutation in BAP1 arose later on the other copy of Chromosome 3 in a tumor subclone, followed by a gain of Chromosome 8q. These findings provide a mechanistic explanation for the well-known clinical association between ocular melanocytosis and UM by showing that this predisposing condition introduces the first "hit" and thereby increases the stochastic likelihood of acquiring further aberrations leading to UM.

Flavoprotein Fluorescence Correlation with Visual Acuity Response in Patients Receiving Anti-VEGF Injection for Diabetic Macular Edema.

Anti-VEGF treatment of diabetic macular edema (DME) complicating diabetic retinopathy (DR) has greatly improved structural and visual outcomes for patients with diabetes mellitus. However, up to 50% of patients are either nonresponsive or refractory to anti-VEGF treatment (no improvement in BCVA or central macular thickness (CMT)). It is believed that factors such as mitochondrial structural and functional damage, due to oxidative stress, are partially responsible for this lack of improvement. Flavoprotein fluorescence (FPF) has been shown to be a sensitive marker of mitochondrial function and has been found to correlate with the degree of diabetic retinopathy. FPF may also provide additional information regarding therapeutic response of patients receiving anti-VEGF treatment for DME. Eight patients with DR and DME with clinically significant DME (CSDME) who underwent anti-VEGF (bevacizumab) treatment were imaged before injection and at follow-up visit using FPF in addition to standard color fundus photography and OCT CMT. A strong correlation r = 0.98 (p = 0.000015) between the FPF decrease and the BCVA improvement was observed; BCVA improved as FPF values decreased. Notably, in the same patients, the correlation between OCT CMT decrease and BCVA improvement (r = 0.688) was not found to be significant (p = 0.13). These findings suggest that FPF can detect improvement in metabolic function preceding structural improvement and even with small changes in edema. Additionally, FPF may be supplementary to current diagnostic methods for earlier detection of therapeutic response to anti-VEGF treatment in patients with DME.

Punctuated evolution of canonical genomic aberrations in uveal melanoma.

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is "set in stone" early in tumor evolution and may explain why advances in primary treatment have not improved survival.