Long-term visual outcomes and fluid compartment changes in limited-early versus early response to anti-VEGF treatment for diabetic macular edema.

OBJECTIVE: This study assessed best visual acuity (BVA) and central subfield thickness (CST) outcomes for LER (limited early responder) and ER (early responder) patients at 24 and 36 months. DESIGN: Retrospective chart review PARTICIPANTS: One-hundred and twelve patients characterized at 3 months after their first anti-VEGF injections as either LER if they met the anatomic criteria (aLER = CST reductions ≤ 10%), visual criteria (vLER = ETDRS letter gains < 5 letter), or both (cLER). All other patients were classified as ER (aER/vER/cER). METHODS: Variables collected include CST and ETDRS letters at baseline, 3, 24, and 36 months following injections, comorbidities, smoking status, demographics, baseline systemic factors, and the type and quantity of anti-VEGF injections. Analyses were performed using Welch's t-test, multivariable linear and multivariable logistic regression. RESULTS: BVA changes from 3 months were significant between cLER versus cER and vLER versus vER groups (p < 0.05). There was a greater decrease in mean BVA from 3 months to 36 months in the cER group compared to the cLER group. Alternatively, mean BVA decreased in the vER cohort, while the vLER cohort slightly increased. CST changes from 3 months were statistically significant (p < 0.01) between all LER and ER groups with LER groups showing greater reductions compared to ER counterparts. BVA and CST changes from baseline to 24 and 36 months were not significant after controlling for baseline differences between LER and ER groups. CONCLUSION: Results highlight the value of long-term anti-VEGF treatment and the need to further explore options that may lead to continued BVA improvements beyond 3 months.

Correlation of limited-early-response status with 12-month CST, BVA, and machine learning-quantified retinal fluid in diabetic macular oedema in routine clinical practice.

BACKGROUND/OBJECTIVES: Anti-VEGF treatment response in DMO has been measured by changes in the central subfield thickness (CST) and best visual acuity (BVA) outcomes at 3 months after initial treatment, termed early or limited early response (ER/LER). This study correlates LER with 12-month BVA, CST, and retinal fluid volumes quantified by a machine learning algorithm on optical coherence tomography (OCT). SUBJECTS/METHODS: The study included treatment naïve DMO patients ≥ 18 years with OCT scans at baseline (M0), M3, M6, and M12. The 220 patients were categorized as limited early responders (LER) if they had ≤ 10% CST reduction and/or < 5 ETDRS letter gain at M3. BVA, CST, and subretinal (SRF), intraretinal (IRF), and total retinal (TRF) fluid volumes quantified by a machine learning algorithm were compared between groups and across time. RESULTS: At M12, the anatomic LER (aLER), defined solely by CST, had significantly worse BVA and CST versus the anatomic ER (aER) group (p < 0.001). Retinal fluid M12 outcomes did not significantly vary between all LER and ER groups. No significant BVA, CST, TRF, and IRF variance across time for LER was found (p > 0.1). CONCLUSIONS: BVA and CST M12 outcomes vary by aLER/aER status indicating that CST may be a strong predictor of treatment outcomes, while retinal fluid volumes were not predicted by LER status.

Real-world effects of anti-vascular endothelial growth factor injection frequency on visual outcomes in patients with diabetic macular oedema.

BACKGROUND AND OBJECTIVE: Anti-vascular endothelial growth factor (VEGF) injections are often administered less frequently in real-world treatment of diabetic macular oedema (DMO) than what was studied in clinical trials. This study aims to characterise real-world DMO treatment patterns and the effect of treatment intervals on patient outcomes. STUDY DESIGN/PATIENTS AND METHODS: This was a retrospective study of 291 patients with DMO treated with anti-VEGF therapy. 12- and 24-month best visual acuity (BVA) and central subfield thickness (CST) were compared between injection interval groups, which were determined by averaging the two most recent injection intervals. Multiple linear regressions were performed to identify factors associated with injection interval, BVA, and CST. RESULTS: 48.8% of patients received injections less than or equal to every 8 weeks (≤ q8w), 27.5% between every 8 to 12 weeks (q8-12w), and 23.7% greater than every 12 weeks (> q12w). Baseline CST was similar (p = 0.32), but BVA differed significantly in q8-12w patients (p = 0.0095). BVA and CST at 12 months were similar, but q8-12w patients experienced greater 12-month BVA improvement (7.36 ± 12.4 letters) than > q12w patients (1.26 ± 12.3 letters; p = 0.0056). 24-month BVA and CST changes were similar between groups (p = 0.30 and 0.87). Baseline BVA, HbA1c, and sex were associated with 12-month BVA, and baseline BVA and CST were associated with 12-month CST. CONCLUSION: Many patients experienced improvements in BVA and CST over 12 months of treatment despite receiving less frequent anti-VEGF therapy than recommended in the pivotal trials. The present study showed that extended treatment intervals with bevacizumab were effective in preserving vision of many individuals with high baseline BVA.

Long-Term Outcomes in Early versus Limited Response to Anti-VEGF Treatment for Retinal Vein Occlusion.

OBJECTIVE: Macular edema secondary to retinal vein occlusion (RVO) is a sight-threatening condition. Previous studies showed that early responders (ERs) who respond well to anti-VEGF injections within 3 months of treatment have better outcomes, as measured by best visual acuity (BVA) and central subfield thickness (CST) at 12 months postinjection initiation compared with limited early responders (LERs). This study analyzed whether ER eyes continue to respond better than LER eyes over longer periods. This study also aimed to identify baseline comorbidities associated with response status. DESIGN: Retrospective cohort study. PARTICIPANTS: Patients aged > 18 years with RVO-related macular edema treated with anti-VEGF injections. METHODS: Patients were categorized as ERs or LERs. Limited early responder eyes were defined as having CST reduction < 10%, BVA gain < 5 ETDRS letters, or both at 3 months after anti-VEGF initiation. Best visual acuity and CST changes over the 24- and 36-month period after the first anti-VEGF treatment were compared between ERs and LERs. Patient characteristics and systemic comorbidities were identified by chart review. Statistical analysis involved the Levene test, Welch t test, and Welch analysis of variance. MAIN OUTCOME MEASURES: Best visual acuity and CST changes over the initial 24-month and 36-month periods after treatment. RESULTS: The 24-month cohort included 68 ERs and 39 LERs, and the 36-month cohort included 58 ERs and 33 LERs. At the 24-month time point, there were significant differences in BVA and CST gains between ERs (+19.8 letters, -221.2 um) and LERs (-2.4 letters, -90.1 um; P < 0.001, P < 0.01). Similarly, at 36 months, there were significant differences in BVA and CST gains between ERs (+17.7 letters, -229.3 um) and LERs (+1.3 letters, -128 um; P < 0.001, P < 0.05). After controlling for differences in baseline BVA and CST, only the 24-month change in BVA remained significant (P < 0.001). There were no significant associations between response status and cardiopulmonary, endocrine, and oncologic comorbidities. CONCLUSIONS: Early responder eyes with branched retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) have better functional responses to anti-VEGF injections at 24 months compared with LER eyes, even after controlling for baseline differences. Early identification of eyes as ERs or LERs in BRVO and CRVO may predict long-term functional prognoses. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Mechanism of the Multistep Catalytic Cycle of 6-Hydroxynicotinate 3-Monooxygenase Revealed by Global Kinetic Analysis.

The class A flavoenzyme 6-hydroxynicotinate 3-monooxygenase (NicC) catalyzes a rare decarboxylative hydroxylation reaction in the degradation of nicotinate by aerobic bacteria. While the structure and critical residues involved in catalysis have been reported, the mechanism of this multistep enzyme has yet to be determined. A kinetic understanding of the NicC mechanism would enable comparison to other phenolic hydroxylases and illuminate its bioengineering potential for remediation of N-heterocyclic aromatic compounds. Toward these goals, transient state kinetic analyses by stopped-flow spectrophotometry were utilized to follow rapid changes in flavoenzyme absorbance spectra during all three stages of NicC catalysis: (1) 6-HNA binding; (2) NADH binding and FAD reduction; and (3) O2 binding with C4a-adduct formation, substrate hydroxylation, and FAD regeneration. Global kinetic simulations by numeric integration were used to supplement analytical fitting of time-resolved data and establish a kinetic mechanism. Results indicate that 6-HNA binding is a two-step process that substantially increases the affinity of NicC for NADH and enables the formation of a charge-transfer-complex intermediate to enhance the rate of flavin reduction. Singular value decomposition of the time-resolved spectra during the reaction of the substrate-bound, reduced enzyme with dioxygen provides evidence for the involvement of C4a-hydroperoxy-flavin and C4a-hydroxy-flavin intermediates in NicC catalysis. Global analysis of the full kinetic mechanism suggests that steady-state catalytic turnover is partially limited by substrate hydroxylation and C4a-hydroxy-flavin dehydration to regenerate the flavoenzyme. Insights gleaned from the kinetic model and determined microscopic rate constants provide a fundamental basis for understanding NicC's substrate specificity and reactivity.

Characterizing and Predicting Limited Early Response in Eyes With Retinal Vein Occlusion Using Machine Learning in Routine Clinical Practice.

OBJECTIVE: To characterize and predict limited early responders (LER) in eyes with retinal vein occlusion (RVO) treated with anti-vascular endothelial growth factor (anti-VEGF). PATIENTS AND METHODS: This retrospective cohort study of 116 eyes with edema secondary to RVO characterized early responders (ER) and LER 3 months after initiating anti-VEGF treatment. Baseline characteristics and 12-month outcomes were compared. A machine learning (ML) algorithm was developed to predict LER. RESULTS: At baseline, LER had higher best-corrected visual acuity (BCVA) than ER (P< 0.0001) and lower central subfield thickness (CST) than ER (P< 0.01). At 12 months, change in BCVA was + 0.8 and + 21.4 letters, and change in CST was -104.4 and -187.1 µm for LER and ER, respectively (P < 0.0001, P < 0.05). The ML algorithm achieved area under the receiver operating characteristic curve = 0.73. CONCLUSION: ER eyes experienced greater functional and anatomical improvements at 12 months in routine clinical practice than LER. The ML algorithm achieved moderately high performance predicting LER. [Ophthalmic Surg Lasers Imaging Retina 2023;54:231-237.].

Predictors of limited early response to anti-vascular endothelial growth factor therapy in neovascular age-related macular degeneration with machine learning feature importance.

PURPOSE: Patients with neovascular age-related macular degeneration (nAMD) have varying responses to anti-vascular endothelial growth factor injections. Limited early response (LER) after three monthly loading doses is associated with poor long-term vision outcomes. This study predicts LER in nAMD and uses feature importance analysis to explain how baseline variables influence predicted LER risk. METHODS: Baseline age, best visual acuity (BVA), central subfield thickness (CST), and baseline and 3 months intraretinal fluid (IRF) and subretinal fluid (SRF) for 286 eyes were collected in a retrospective clinical chart review. At month 3, LER was defined as the presence of fluid, while early response (ER) was the absence thereof. Decision tree classification and feature importance methods determined the influence of baseline age, BVA, CST, IRF, and SRF, on predicted LER risk. RESULTS: One hundred and sixty-seven eyes were LERs and 119 were ERs. The algorithm achieved area under the curve = 0.66 in predicting LER. Baseline SRF was most important for predicting LER while age, BVA, CST, and IRF were somewhat less important. Nonlinear trends were observed between baseline variables and predicted LER risk. Zones of increased predicted LER risk were identified, including age <74 years, and CST <290 or >350 µm, IRF >750 nL, and SRF >150 nL. CONCLUSION: These findings explain baseline variable importance for predicting LER and show SRF to be the most important. The nonlinear impact of baseline variables on predicted risk is shown, increasing understanding of LER and aiding clinicians in assessing personalized LER risk.

The Impact of Early Residual Fluid After Anti-Vascular Endothelial Growth Factor Initiation in Patients With Neovascular Age-Related Macular Degeneration: A Meta-Analysis Review.

Fluid in neovascular age-related macular degeneration is often used to assess patient response to anti-vascular endothelial growth factor therapy. Various studies theorize that early residual fluid (ERF), noted as persistence of intraretinal fluid and subretinal fluid after the anti-vascular endothelial growth factor loading phase (LP), may be predictive of visual outcomes. This meta-analysis examined the existing literature on the relationship between ERF and long-term visual acuity (VA) and found that those who were fluid-free after the LP tended to have the highest VA gains overall. Early intraretinal fluid appeared to be associated with reduced VA gains, whereas the impact of early sub-retinal fluid was more debated. For those with ERF, monthly or more frequent dosing regimens following the LP appeared most optimal for VA. As most studies in this review were post hoc analyses, this highlights the need for real-world studies investigating ERF and its effect on visual outcomes in neovascular age-related macular degeneration. [Ophthalmic Surg Lasers Imaging Retina 2022;53:506-513.].

Characterizing Early Residual Fluid in Neovascular Age-Related Macular Degeneration using Machine Learning in Routine Clinical Practice.

PURPOSE: Neovascular age-related macular degeneration (nAMD) often requires intensive therapy with anti-VEGF injections. In prior post hoc studies, early residual fluid (ERF) after the loading phase was associated with poorer treatment outcomes. This retrospective study examined the impact of ERF on vision using machine learning (ML) methods in routine clinical practice. DESIGN: Retrospective cohort. PARTICIPANTS: This study included treatment-naïve patients with nAMD who were initiated on anti-VEGF between 2012 and 2018, with at least 1 year of follow-up. METHODS: Overall, 286 patients with nAMD were included. An ML algorithm quantified intraretinal fluid (IRF), subretinal fluid (SRF), and total retinal fluid from OCTs. The ERF group included those with fluid at week 12 and was further stratified by fluid subtype. Paired t tests and analysis of variance compared best visual acuity (BVA) and fluid among subgroups, and a quartile analysis correlated fluid volumes to week 52 BVA. The risk of ERF was predicted from baseline factors using 3 ML methods: Ridge logistic regression, k nearest neighbors classification, and support vector classification. MAIN OUTCOME MEASURES: Mean change in BVA from baseline to week 52 according to week 12 fluid status. RESULTS: At week 12, 58.4% of patients had ERF. The breakdown of those in the ERF group included SRF-only (45.5%), IRF-only (21.6%), and IRF and SRF (32.9%). The ERF and ERF-free groups had similar BVA gains from baseline to week 52 (+5.7 ± 15.4 vs. +4.9 ± 18; P = 0.69). Examining specific ERF subgroups revealed no significant differences among the IRF-only (+4.6 ± 16.4), SRF-only (+5.6 ± 12.5), and IRF and SRF (+6.6 ± 18.5, P = 0.93) groups. Quartile analysis of week 12 fluid revealed no predictive pattern for BVA gains. Three ML methods were developed to predict those at risk for ERF achieved equivalent performance, with F1 score of 0.73 to 0.76. CONCLUSIONS: These results diverge from prior post hoc studies, in that there was no significant difference in long-term BVA gains between ERF and ERF-free cohorts, as well as between the week 12 fluid subgroups.

Mechanism of 6-Hydroxynicotinate 3-Monooxygenase, a Flavin-Dependent Decarboxylative Hydroxylase Involved in Bacterial Nicotinic Acid Degradation.

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K., et al. (2016) Biochemistry 55, 3432-3446]. Results with Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004) and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and Kd6HNA analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, and Y225F) identify Tyr215 and His47 as critical determinants both of 6-HNA binding ( KdY215F/ KdWT > 240; KdH47E/ KdWT > 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses are in accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.