Standardization and clinical applications of retinal imaging biomarkers for cardiovascular disease: a Roadmap from an NHLBI workshop.

The accessibility of the retina with the use of non-invasive and relatively low-cost ophthalmic imaging techniques and analytics provides a unique opportunity to improve the detection, diagnosis and monitoring of systemic diseases. The National Heart, Lung, and Blood Institute conducted a workshop in October 2022 to examine this concept. On the basis of the discussions at that workshop, this Roadmap describes current knowledge gaps and new research opportunities to evaluate the relationships between the eye (in particular, retinal biomarkers) and the risk of cardiovascular diseases, including coronary artery disease, heart failure, stroke, hypertension and vascular dementia. Identified gaps include the need to simplify and standardize the capture of high-quality images of the eye by non-ophthalmic health workers and to conduct longitudinal studies using multidisciplinary networks of diverse at-risk populations with improved implementation and methods to protect participant and dataset privacy. Other gaps include improving the measurement of structural and functional retinal biomarkers, determining the relationship between microvascular and macrovascular risk factors, improving multimodal imaging 'pipelines', and integrating advanced imaging with 'omics', lifestyle factors, primary care data and radiological reports, by using artificial intelligence technology to improve the identification of individual-level risk. Future research on retinal microvascular disease and retinal biomarkers might additionally provide insights into the temporal development of microvascular disease across other systemic vascular beds.

Retinal Ischemic Perivascular Lesions Are Associated With Myocardial Infarction in Patients With Coronary Artery Disease.

PURPOSE: To evaluate the association of retinal ischemic perivascular lesions (RIPLs) with myocardial infarction (MI) among patients diagnosed with coronary artery diseases (CAD). DESIGN: Retrospective cross-sectional study. METHODS: Consecutive patients (317 patients) with CAD who underwent macular spectral domain optical coherence tomography (SD-OCT) were captured. Patients with CAD who developed MI were compared to those without MI. SD-OCT were reviewed by 2 independent and masked graders for the presence of RIPLs. Medical records were reviewed. Multivariate logistic regression analysis was used to evaluate the relationship between RIPLs and MI including the following covariates age, gender, smoking status, hypertension, diabetes, dyslipidemia and body mass index. RESULTS: Of 317 patients with CAD for whom OCT scans were available to study, there were 54 (17%) with a history of MI. A higher prevalence of RIPLs was observed in the MI group compared to the non-MI group (59.3% vs 35.7%; P < .001). The presence of RIPLs was significantly associated with MI with an odds ratio of 3 (1.91-4.74; P < .001), after adjusting for age, gender, smoking status, hypertension, diabetes, dyslipidemia, and body mass index. CONCLUSIONS: The presence of RIPLs, detected with SD-OCT, is significantly associated with MI in patients with CAD. These findings underscore the potential clinical utility of incorporating RIPL evaluation in the medical management of CAD.

Retinal Ischemic Perivascular Lesions in Individuals With Atrial Fibrillation.

Background We previously demonstrated that retinal ischemic perivascular lesions (RIPLs), which are indicative of ischemia in the middle retina, may be a biomarker of ischemic cardiovascular disease. In this study, we sought to determine the relationship between RIPLs and atrial fibrillation, a common source of cardiac emboli. Methods and Results In this case-control study, we identified individuals between the ages of 50 and 90 years who had undergone macular spectral domain optical coherence tomography imaging. Individuals with atrial fibrillation were identified, and age- and sex-matched individuals from the same pool, but without a diagnosis of atrial fibrillation, were selected as controls. Spectral domain optical coherence tomography scans were reviewed by 3 independent and masked observers for presence of RIPLs. The relationship between RIPLs and atrial fibrillation was analyzed using multivariable logistic regression models. There were 106 and 91 subjects with and without atrial fibrillation, respectively. The percentage of subjects with RIPLs was higher in the atrial fibrillation group compared with the control group (57.5% versus 37.4%; P=0.005). After adjusting for age, sex, smoking history, hypertension, diabetes, coronary artery disease, carotid stenosis, stroke, and myocardial infarction, the presence of RIPLs was significantly associated with atrial fibrillation, with an odds ratio of 1.91 (95% CI, 1.01-3.59). Conclusions RIPLs are significantly associated with atrial fibrillation, independent of underlying ischemic heart disease or cardiovascular risk factors. This association may inform the diagnostic cardiovascular workup for individuals with RIPLs incidentally detected on optical coherence tomography scan of the macula.

Determinants of overall survival in patients with metastatic uveal melanoma.

BACKGROUND: Despite improvements in the treatment of primary uveal melanoma (UM), patients with metastatic disease continue to exhibit poor survival. METHODS: A retrospective review of metastatic UM patients at Yale (initial cohort) and Memorial Sloan Kettering (validation cohort) was conducted. Cox proportional hazards regression was used to determine baseline factors that are associated with overall survival, including sex, Eastern Cooperative Oncology Group (ECOG) Performance Status Scale, laboratory measurements, metastasis location, and use of anti-CTLA-4 and anti-PD-1 therapies. Differences in overall survival were analyzed using Kaplan-Meier analysis. RESULTS: A total of 89 patients with metastatic UM were identified; 71 and 18, in the initial and validation cohorts, respectively. In the initial cohort, median follow-up was 19.8 months (range, 2-127 months) and median overall survival was 21.8 months (95% CI, 16.6-31.3). Female sex, anti-CTLA-4, and anti-PD-1 therapy were associated with better survival outcomes with adjusted death hazard ratios (HRs) of 0.40 (95% CI, 0.20-0.78), 0.44 (0.20-0.97), and 0.42 (0.22-0.84), respectively, whereas development of hepatic metastases and ECOG score ≥1 (per 1 U/L) were associated with worse survival outcomes with HRs of 2.86 (1.28-7.13) and 2.84 (1.29-6.09), respectively. In both the initial and validation cohorts, use of immune checkpoint inhibitors was associated with improved overall survival after adjusting for sex and ECOG score, with death HRs of 0.22 (0.08-0.56) and 0.04 (0.002-0.26), respectively. CONCLUSIONS: Development of extrahepatic-only metastases, ECOG of 0, immune checkpoint therapy, and female sex were each associated with more than 2-fold reductions in risk of death. PLAIN LANGUAGE SUMMARY: Metastatic uveal melanoma patients face limited treatment options and poor survival rates. Results from this retrospective analysis indicate that immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1 therapies, were associated with improved survival outcomes. Factors such as extrahepatic-only metastases, better baseline performance status, and female sex contributed to a more than 2-fold reduction in death risk. These findings highlight the potential of immunotherapy in treating metastatic uveal melanoma.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Validation of the Prognostic Usefulness of the Gene Expression Profiling Test in Patients with Uveal Melanoma.

PURPOSE: To validate the prognostic usefulness of gene expression profile (GEP) testing in patients with uveal melanoma. To determine whether combining tumor size with the GEP classification provides additional prognostic value. DESIGN: Retrospective analysis. PARTICIPANTS: Patients with a diagnosis of choroidal melanoma examined at Yale New Haven Hospital; University of California, San Diego; and Memorial Sloan Kettering Cancer Center. METHODS: Patients' demographic and clinical data and tumor characteristics were collected. Univariate and multivariate Cox hazard regression analysis were used to assess the association between tumor characteristics and GEP classification with metastasis as an outcome. MAIN OUTCOME MEASURES: Metastasis-free survival (MFS). RESULTS: Of the 337 individuals included in the study, 87 demonstrated metastases. The mean follow-up time was 37.2 (standard deviation [SD], 40.2) months for patients with metastases and 55.0 (SD, 49.3) months for those without metastases. Tumors of larger thickness and GEP class 2 (vs. class 1) were associated significantly with increased risk of metastasis. Tumor thickness showed better prognostic usefulness than GEP classification (Wald statistic, 40.7 and 24.2, respectively). Class 2 tumors with a thickness of 7.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 7.0 mm (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.61-6.51), whereas class 1 tumors with a thickness of 9.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 9.0 mm (HR, 2.07; 95% CI, 0.86-4.99). No difference in MFS was found between patients with class 1A tumors compared with those with class 1B tumors (P = 0.8). Patients with class 2 tumors showed an observed 5-year MFS of 47.5% (95% CI, 36.0%-62.8%). CONCLUSIONS: Tumor size was the most significant predictor of metastasis and provided additional prognostic value independent of GEP classification. In addition, rates of metastasis for class 2 tumors were lower than estimates reported by Castle Bioscience, and no difference in rates of metastasis were found between class 1A and 1B tumors. This indicates that tumor size should be accounted for when relying on GEP for prognostication and that patients with GEP class 1A or 1B tumors may benefit from the same metastatic surveillance protocols. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Retinal vein occlusion is associated with stroke independent of underlying cardiovascular disease.

BACKGROUND: Stroke is a leading cause of mortality and morbidity. Thus, identifying associated risk factors may lead to earlier interventions aimed at reducing the risk of stroke development. Since cardiovascular disease simultaneously increases the risk of stroke and retinal vein occlusion (RVO), we sought to determine whether RVO is associated with the risk of stroke independent of underlying cardiovascular co-morbidities. METHODS: In this cross-sectional study, we reviewed the records of 80,754 individuals who were evaluated by an ophthalmologist over a 6-year period. We identified individuals with RVO, stroke and cardiovascular diseases including hypertension, diabetes mellitus, carotid disease, coronary artery disease and atrial fibrillation. Multivariable logistic regression models were used to analyze odds ratios for RVO and stroke. RESULTS: After adjusting for age, sex, cardiovascular disease and other risk factors, we found that the presence of RVO was associated with an odds ratio for stroke of 1.73 (CI, 1.40-2.12, p < 0.001). The association between RVO and stroke, after adjusting for sex and cardiovascular co-morbidities, was significantly stronger in individuals younger than 50 years of age, with an odds ratio of having a stroke of 3.06 (1.34-6.25, p < 0.001), while the presence of RVO in individuals older than 85 years was not significantly associated with stroke 1.19 (0.77-1.79, p = 0.41). CONCLUSIONS: Our findings demonstrate that RVO is significantly associated with stroke, even after adjusting for underlying cardiovascular co-morbidities. This association was highly significant in younger subjects, while not significant in older individuals.

BAP1 methylation: a prognostic marker of uveal melanoma metastasis.

Uveal melanoma, the most common intraocular primary cancer in adults, is characterized by striking variability in metastatic tendencies. BAP1 deletion in the primary tumor is associated with uveal melanoma metastasis, but it cannot always be resolved by bulk DNA sequencing of heterogeneous tumors. Here, we show that assessment of BAP1 methylation is an accurate and readily clinically actionable assay to accurately identify high-risk uveal melanoma patients.

Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression.

Chromosomal instability (CIN) and epigenetic alterations have been implicated in tumor progression and metastasis; yet how these two hallmarks of cancer are related remains poorly understood. By integrating genetic, epigenetic, and functional analyses at the single cell level, we show that progression of uveal melanoma (UM), the most common intraocular primary cancer in adults, is driven by loss of Polycomb Repressive Complex 1 (PRC1) in a subpopulation of tumor cells. This leads to transcriptional de-repression of PRC1-target genes and mitotic chromosome segregation errors. Ensuing CIN leads to the formation of rupture-prone micronuclei, exposing genomic double-stranded DNA (dsDNA) to the cytosol. This provokes tumor cell-intrinsic inflammatory signaling, mediated by aberrant activation of the cGAS-STING pathway. PRC1 inhibition promotes nuclear enlargement, induces a transcriptional response that is associated with significantly worse patient survival and clinical outcomes, and enhances migration that is rescued upon pharmacologic inhibition of CIN or STING. Thus, deregulation of PRC1 can promote tumor progression by inducing CIN and represents an opportunity for early therapeutic intervention.

Prevalence of subclinical retinal ischemia in patients with cardiovascular disease - a hypothesis driven study.

BACKGROUND: Cardiovascular disease is the leading cause of mortality and disability worldwide. A noninvasive test that can detect underlying cardiovascular disease has the potential to identify patients at risk prior to the occurrence of adverse cardiovascular events. We sought to determine whether an easily observed imaging finding indicative of retinal ischemia, which we term 'retinal ischemic perivascular lesions' (RIPLs), could serve as a biomarker for cardiovascular disease. METHODS: We reviewed optical coherence tomography (OCT) scans of individuals, with no underlying retinal pathology, obtained at UC San Diego Health from July 2014 to July 2019. We identified 84 patients with documented cardiovascular disease and 76 healthy controls. OCT scans were assessed for evidence of RIPLs. In addition, the 10-year atherosclerotic cardiovascular disease (ASCVD) risk calculator was used to risk-stratify the subjects into four different categories. FINDINGS: Patients with documented cardiovascular disease had higher number of RIPLs compared to healthy controls (2.8 vs 0.8, p < 0.001). After adjusting for age, sex, smoking history, systolic blood pressure and triglycerides, cholesterol and hemoglobin A1C levels, each RIPL was associated with an odds ratio of having cardiovascular disease of 1·60 (1.09-2>37). The number of RIPLs in individuals with intermediate and high 10-year ASCVD risk scores was higher than in those with low ASCVD risk scores (1.7 vs 0.64, p = 0.02 and 2.9 vs 0.64, p 0.002, respectively). INTERPRETATION: The presence of RIPLs, which are anatomical markers of prior retinal ischemic infarcts, is suggestive of coexisting cardiovascular disease. RIPLs detection, obtained from routine retinal scans, may thus provide an additional biomarker to identify patients at risk of developing adverse cardiovascular events. FUNDING: None.

A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

Relationship between Retinal Vascular Occlusions and Cognitive Dementia in a Large Cross-Sectional Cohort.

PURPOSE: To examine the association between cognitive dementia and retinal vascular occlusions. DESIGN: A retrospective, cross-sectional study. METHODS: Single-institution study population: we reviewed the electronic medical records of 37,208 individuals older than 65 years of age who were evaluated by an ophthalmologist or an optometrist and who also had a medical visit to our institution over a 6-year period. Individuals with and without retinal vascular occlusions were identified by International Classification of Diseases, version 10 (ICD-10) diagnostic codes. MAIN OUTCOME: we analyzed the association between dementia and retinal vascular occlusions after adjusting for covariates which included age, sex, stroke, diabetes mellitus, and hypertension using multiple logistic regression analyses. RESULTS: Compared to subjects without retinal vascular occlusions, those with retinal vascular occlusions had a higher prevalence of dementia (6.7% vs. 9.3%, respectively; P < .001). After adjusting for either age or stroke, there were no significant associations between retinal vascular occlusions and dementia. CONCLUSIONS: Individuals with retinal vascular occlusions have a higher prevalence of dementia. However, this association is secondary to shared underlying risk factors in this population, such as older age and stroke.

PREVALENCE OF MISMATCH REPAIR GENE MUTATIONS IN UVEAL MELANOMA.

PURPOSE: Uveal melanomas are associated with characteristic genetic changes. Germline mutations in mismatch repair (MMR) genes and microsatellite instability have been implicated in the development of numerous malignant neoplasms such as colon and ovarian cancers. The frequency of MMR defects in uveal melanomas has yet to be determined. METHODS: Here, we analyzed the frequency of MMR gene mutations in uveal melanoma specimens from the University of California, San Diego (UCSD), The Cancer Genome Atlas (TGCA), and the Catalogue of Somatic Mutations in Cancer (COSMIC). RESULTS: We identified only two mutations in a MMR gene: one premature stop codon in the PMS gene within the UCSD cohort (0.5% frequency) and one in-frame deletion in MSH3 within the COSMIC database (0.8% frequency). We report copy number variation of MLH1 in monosomy 3 and show decreased mRNA expression of MLH1 in uveal melanoma specimens with monosomy 3. Expression levels of MLH1 were not found to correlate with the observed number of total mutations. CONCLUSION: Overall, we show that mutations in MMR genes in uveal melanoma specimens are exceedingly rare, and although one copy of MLH1 is lost in monosomy 3, it does not seem to have pathologic consequences in uveal melanoma pathogenesis.

Molecular Characteristics of Uveal Melanoma: Insights from the Cancer Genome Atlas (TCGA) Project.

The Cancer Genome Atlas (TCGA) uveal melanoma project was a comprehensive multi-platform deep molecular investigation of 80 uveal melanoma primary tissue samples supported by the National Cancer Institute. In addition to identification of important mutations for the first time, it identified four different clusters (subgroups) of patients paralleling prognosis. The findings of the TCGA marker paper are summarized in this review manuscript and other investigations that have stemmed from the findings of the TCGA project are reviewed.

Paracentral Acute Middle Maculopathy and the Ischemic Cascade Associated With Retinal Vascular Occlusion.

PURPOSE: To analyze the spectrum of ischemia associated with paracentral acute middle maculopathy (PAMM) in eyes with acute retinal vascular occlusion and to describe an ischemic cascade. DESIGN: A retrospective observational case series. METHODS: Patients presenting with PAMM secondary to acute retinal vascular occlusion were identified. Analysis of multimodal imaging was performed at baseline and at follow-up visits to elucidate the patterns and progression of ischemia within the retinal layers. RESULTS: Multimodal retinal imaging from 16 eyes of 16 patients with acute retinal vascular occlusion associated with PAMM was studied. Analysis of en face optical coherence tomography (OCT) segmentation of the inner nuclear layer (INL) identified distinct patterns of PAMM correlating with the severity of ischemia and not the type of occlusion. A perivenular fern-like PAMM pattern was associated with better visual outcomes (average final visual acuity was 20/25). This pattern was noted to sequentially progress in 2 cases to a diffuse globular PAMM pattern in the INL, or to a pattern of ischemia involving both the middle and inner retinal layers with commensurate vision loss. Globular patterns of PAMM or ischemia involving both the middle and inner retina correlated with poorer visual outcomes (average final visual acuity was counting fingers at 5.5 ft). These various patterns of ischemia developed in eyes with retinal vascular occlusions in which blood flow through the retinal capillary plexuses was present but was significantly reduced and delayed. CONCLUSIONS: This study describes OCT findings suggestive of an ischemic cascade in eyes with retinal vascular occlusion. The middle retina at the level of the deep capillary plexus, especially at the venular pole, may be more vulnerable to ischemic injury.