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Dr.Pallavi Sarate

Presenting author :Dr Pallavi Sarate , MS,DNB,FMRF(VR),FCPS,DOMS,Consultant & Surgeon in Vitreoretina , Lotus eye hospital, Juhu,Mumbai

ABSTRACT :

PURPOSE:

To determine the role of OCT biomarkers such as subfoveal photoreceptor outer segment (PROS) length and thickness , integrity of ellipsoid zone(EZ), External limiting membrane (ELM) and  visual outcome after bevacizumab for DME

METHODS:

A retrospective study of 42 treatment naive  eyes with fovea  involving DME  .Subfoveal OCT scans were performed prior to , 45 days (6 weeks)  and 3 months post treatment. Biomarkers assessed were changes in the thickness and continuity of the PROS subfoveally, integrity of EZ and ELM , presence of sub retinal fluid, outer nuclear layer cysts, Inner nuclear layer cysts, subfoveal External limiting membrane continuity,subfoveal ellipsoid zone continuity , subfoveal PROS thickness, High reflective foci location. Relationship of each parameter was compared with visual acuity

RESULTS :

Mean follow-up was 12.8±11.5 months. In 36 of the 42 eyes, DME resolved.Statistical analyses showed that the pretreatment PROS thickness & integrity of EZ and ELM  had the highest correlation with the post treatment best-corrected visual acuity (P<0.050).

Introduction :

Vision loss associated with diabetic retinopathy is most commonly caused by diabetic macular edema (DME), which affects approximately 7% of all diabetic patients .(19)

Breakdown of the blood–retina barrier and consequent leakage of abnormal fluid into the retinal tissuedue to vascular endothelial growth factor (VEGF) release are understood to be the major factor in the development of DME. Consequently, intravitreal administration of anti- VEGF drugs is considered to be the most efficient therapy for DME resolution and visual acuity improvement (18)

Anatomic measures on spectral-domain (SD) OCT, such as precise evaluation of individual layers, qualitative assessment of fluid distribution and existence of hyper- reflective foci (HRF), continuity of External limiting membrane (ELM), integrity of ellipsoid zone (EZ), thickness of photoreceptor outer segment length (PROS) could predict treatment success or failure to different therapies in term of visual improvement.

The purpose of this study was to investigate whether characteristics identified on SD OCT may serve as biomarkers and predict treatment response  to bevacizumab in terms of visual outcome in patients with DME.

Key words: Diabetic macular edema, anti-VEGF, OCT biomarkers

Methods :

This retrospective study ,comprises of Diabetic macular edema patients , treated with Intravitreal bevacizumab patients  from January 20, 2014, to March 20 ,2018

Naive patients with DME , treated with ranibizumab injections for DME were included in the study fulfilling following inclusion and exclusion criteria

Inclusion Criteria

  • Age above 35 years;
  • Type 2 diabetes mellitus;
  • DME (treatment naïve eyes ) causing visual loss, with study eye BCVA 6/12 to 6/60 on Snellens chart , macular edema diagnosed clinically and by retinal thickness of >300 mm in the central subfield; and intraretinal or subretinal fluid (SRF) seen on SD OCT;

Exclusion Criteria

  • Another concomitant ocular disease that causes macular edema (i.e., neovascular age-related macular degeneration or choroidal neovascularization due to other reasons, retinal vein occlusion, uveitis, and recent intraocular surgery possibly causing postsurgical macular edema);except for the presence of cataract
  • Previous treatment with intraocular corticosteroids ,laser or anti VEGF injections

(4) Presence of proliferative diabetic retinopathy;

(5) Previous vitrectomy surgery; and

6) Other chorioretinal diseases like central serous chorioretinopathy, high myopia, chorioretinitis, or any other fundus disease associated with morphological or functional changes.

7) Eyes with significant media opacities which can result in poor OCT signal were excluded

Optical Coherence Tomography Analysis

SD-OCT scans were performed on all eyes during each session using Cirrus 500 OCT. A prototype algorithm was developed for quantitative assessment of various parameters mentioned in Table which were calculated for three parameters; macular grid (6mm × 6mm), central subfield (1mm), and center foveal point (0.33mm).A total of three “high-quality” scans were obtained; these were defined as scans with a signal strength ≥6 .All scans were performed by the same certified OCT technician and  were recorded at baseline and at 45 days (6 weeks ) and 3 months after Bevacizumab. Visual acuity was checked on Snellens chart.

Qualitative and quantitative evaluations of SD OCT images performed at baseline assessed the presence of several morphologic features including

  • Subretinal fluid (SRF );
  • Cystoid changes in the outer nuclear layer (ONL)
  • Presence of cystoid changes in the inner nuclear layer (INL);
  • Continuity of the External limiting membrane (ELM)layer  (completely continuous, partly disrupted, completely disrupted);
  • Subfoveal Ellipsoid zone continuity (completely continuous, partly disrupted, completely disrupted);
  • Subfoveal Photoreceptor outer segment (PROS ) thickness
  • Presence of HRF, (subfoveal and extrafoveal ) .                                                        The listed features were evaluated on 3 horizontal OCT scans: 1 b-scan encompassing the fovea, 2 b-scans respectively 500 mm superior and 500 mm inferior to the fovea. Grading of the OCT images was performed by single retina specialists who was blinded to the functional and anatomic results.

Mean follow-up was 12.8±11.5 months.

Statistical analysis and Optical Coherence Tomography Predictors for Treatment Response

The outcome measures (BCVA response at baseline, 45 days and 3 months post bevacizumab injection ) were run by testing the following predictors at baseline: (1) BCVA( gain >10 letters, gain < 9 letters , loss >10 letters; (2) the presence of SRF; (3) the presence of ONL cysts; (4) presence of INL cysts; (5) Subfoveal ELM continuity ; (6) Ellipsoid zone continuity; (7 )PROS length (11) HRF location. Chi square test was applied to calculate P value and to analyse statistically significant variables.

Results :

A total of 42 eyes from 58 patients were included in the analysis.

24 eyes (57%)were phakic and 18 (42.8%) were pseudophakic.

Statistically significant parameters which were found during this study are as follows.

Image add

Subfoveal external limiting membrane continuity was seen to have statistically significant  (P <0.05%) better  visual outcome at the end of 6 weeks and 3 months post bevacizumab injection. Completely disrupted ELMs were associated with poor visual outcome.( Table 1 and 2 ). BCVA gain >10 letters was seen in 14 out of 18 eyes  (77.7%)with Completely continuous subfoveal  ELMs( P <0.05%) at the end of 6 weeks .( Table 1 )At 3 months follow up post bevacizumab, 12 (75%)out of 16 patients with continuous subfoveal ELM   presented with BCVA gain of >10 letters on ETDRS (P<0.001 %)(Table 2 )

Similarly Ellipsoid zone continuity was seen to be associated with better visual outcome. 16 out of 20 eyes (80%) were associated with >10 letter BCVA gain(P <0.021%) at the end of 6 weeks( Table 1 ).  3 months post bevacizumab , 14/18 patients (77%) showed BCVA gain of > 10 letters (P <0.001%), and 8/10 patients with completely disrupted subfoveal EZ showed > 10 letters BCVA loss.(Table 2 )

13 out of 16 eyes with Photoreceptor outer segment length >21-35 micron showed > 10 letter BCVA gain (P < 0.001 %)at the end 6 weeks.( Table 1 ).After 3 months, out of 14 patients 11 patients (78.5%)showed BCVA improvement of >10 letters (P<0.001%)(Table 2)

Subfoveal High reflective foci (HRF)were associated with poor visual outcome. With Subfoveal HRF 8 out of 10 patients (80%) were associated with > 10 letter loss of BCVA (P < 0.011%) at the end of 6 weeks.( Table 1 )

At 6 weeks followup post bevacizumab, we found 8 patients out of 10(80 %) ( P<0.001) patients with subfoveal HRF had >10 letters BCVA loss. At 3 months follow up 7/12 patients had BCVA loss of > 10 letters( P<0.012).(Table 2 )

Thus statistical analyses showed that the pretreatment PROS thickness & integrity of EZ and ELM  had the highest correlation with the post treatment best-corrected visual acuity (P<0.050).

Discussion :

Diabetic macular edema (DME) is a leading cause of vision loss in working-age adults. Within the past few years, intravitreal vascular endothelial growth factor (VEGF) inhibitors have emerged as preferred therapy for many patients with DME.

Vascular endothelial growth factor (VEGF) has multiple pathological and physiological effects, i.e., angiogenesis, vascular hyperpermeability, induction of vascular changes.(12,13)

Bevacizumab (Avastin; Genentech)( Anti VEGF agent ) is  widely used for the treatment of DME; a formulation approved for systemic cancer therapy is usually repackaged (compounded) in syringes for off-label ophthalmic use and is available at lower cost. (21)

Optical coherence tomography (OCT) is a reliable, quick, sensitive, non-invasive, user-friendly device that provides high-resolution in vivo imaging of retinal microstructures.

“Biomarker”, a merged word of “biological marker”, refers to signs that objectively indicate the state of health, and well-being. These can be anatomical, biochemical, molecular parameters or imaging features. (3)

SD-OCT based biomarkers help us to identify the ultrastructural alterations in retina even in early phases of the disease and their gradation increases with severity of DR. These biomarkers are also useful to evaluate the response to therapy and modify our treatment protocol accordingly.

In the present work, we tested the possibility of using a noninvasive imaging technique SD OCT, in naive patients with DME that have clinical indication for anti-VEGF treatment, to correlate the OCT biomarkers with BCVA outcome in the initial stages of anti-VEGF therapy response to evaluate its predictive value as a prognostic biomarker in the management of DME treatment with anti-VEGF.

Although serous macular detachment (SMD) may be difficult to diagnose by slit-lamp ,biomicroscopy or angiography, SD OCT enables detection of even very subtle SMD .The pathogenesis of submacular fluid in DME remains poorly defined. Apparently, higher levels of vascular leakage from the macular vasculature play a major role in its development and persistence, in addition to vitreomacular traction. (1,2 ) .

According to report by Alasil T el al, the presence of SMD does not seem to correlate with VA in DME. (22)

In our study we did not find statistically significant difference in visual outcome with presence of subretinal fluid post bevacizumab treatment .

Lee et al.reported that outer plexiform (OPL) disruption in SD-OCT corresponded well with the extent of deep capillary plexus loss in eyes with DME and might be useful predictors of response to anti-VEGF treatment. (11,13)

Cystoid changes beneath the outer plexiform layer have been shown to present risk to photoreceptor cells and to impair ellipsoid zone (EZ) integrity (23).In our study, neither the size nor the location of intraretinal cysts altered functional outcome. Apparently, more than the absolute cyst size and volume, the remaining tissue between cysts in the central macula seems to be crucial for good VA.We did not analyze this parameter, and further investigation might provide more information about its impact.

The ELM is the junctional complex between the Müller and photoreceptor cells and has barrier properties against macromolecules . The disrupted ELM might allow blood constituents to pour into the subretinal spaces and damage the photoreceptors, or vice versa.Photoreceptor damage might lead to disruption of the ELM in DME(16)

Two possible explanations should be considered, i.e., damage in the Müller cells might contribute to both ELM disruption and inner retinal thickening, and the disrupted barrier properties in the ELM might dysregulate the fluid dynamics and concomitantly allow accumulation of intraretinal or subretinal fluids (18)

In our study ,at 6 weeks follow up post bevacizumab ,14 out of 18 patients (77.7%) with completely continuous subfoveal ELM showed with BCVA gain >10 letters, and 5 (71.4%) out of 7 patients showed > 10 letter  BCVA loss (P <0.005%) At 3 months follow up post bevacizumab, 12 (75%)out of 16 patients with continuous subfoveal ELM   presented with BCVA gain of >10 letters on Snellens (P<0.001 %), and 7 out of 7 patients (77.7%) with completely disrupted ELM subfoveally presented with> 1o letters of BCVA loss.

The Ellipsoid zone (EZ), previously known as the third high-reflectance band or the junction between the inner and outer segments (IS/OS), clinically represents the photoreceptor integrity in several diseases. The biological EZ is comprised mainly of mitochondria and enables higher levels of energy consumption in the photoreceptors. The absence of the EZ on SD-OCT images might correspond to the anatomic absence or the reduced OCT reflectivity of the EZ. This led us to hypothesize that the mitochondrial dysfunction in the foveal photoreceptors leads to reduced VA in DME.(14,15)

Although it remains ill-defined what blood constituents exacerbate photoreceptor damage, anti-VEGF treatment might block their extravasation with concomitant restoration of the EZ and outer segments on SD-OCT images(16,17).In addition, anti-VEGF therapy might resolve foveal cystoid spaces, which seem to compress and deform the photoreceptor layers

In current study, 16/20 patients (80 %) with completely continuous subfoveal EZ showed > 10 letter gain on Sentinels chart (P<0.021%) 6 weeks post bevacizumab injection.At 3 months post bevacizumab , 14/18 patients (77%) showed BCVA gain of > 10 letters (P <0.001%), and 8/10 patients with completely disrupted subfoveal EZ showed > 10 letters BCVA loss.

With the use of Cirrus HD-OCT 500 and a prototype software algorithm, we have quantified PROS length in patients with DME. Furthermore, we report a relatively strong correlation between  Photoreceptor outer segment (PROS) length and visual acuity in these patients.

Recently, an OCT-based approach has demonstrated a mean cone OS length of 40.6 μm in the fovea of healthy subjects (5).In our study we found it to be 32+/- 5microns.

The modest correlation between retinal thickness and visual acuity that has been previously reported suggests that these thickness measurements are not particularly predictive of visual acuity. We found PROS length measurements correlated more strongly with visual acuity than macular thickness measurement, suggesting that PROS length may represent a novel and reliable correlate of visual acuity in patients with DME. However, PROS length cannot explain all of the variation in visual acuity and other factors like macular ischaemia, retinal cell function, may be important as well.

The stronger correlation of PROS length with visual acuity suggests that the PROS measures may be more directly related to visual function. Photoreceptor outer segment length may be a useful physiologic outcome measure, both clinically and as a direct assessment of treatment effects.(4)

In our study, according to PROS thickness 3 subsets were made PROS 21 -35 microns, 10-20 microns and less than 10 microns.

After 6 weeks,13 out of 16 patients 81%) with subfoveal PROS thickness 21-35 microns showed BCVA gain >10 letters (P<0.001%), and 8 out of 11 patients (72.7%) with PROS thickness < 10 microns showed BCVA loss of > 10 letters( Table 1 ). After 3 months, out of 14 patients 11 patients (78.5%)showed BCVA improvement of >10 letters (P<0.001%) and 8 out of 10 patients (80%) with PROS thickness < 10 microns showed > 10 letters loss(Table 2)

The hyperreflective dots (HRDs) on SD-OCT are well- circumscribed particles that are 20 to 40 lm in diameter, and are of equal or higher reflectivity than the RPE band (6,7). The location of HRDs might be a good indicator that predict the treatment response to anti-VEGF injections.The number of HRDs within an area of 1500 micron radius centered on the fovea on the fovea-spanning horizontal raster scan was manually counted (8)

The origin of HRDs is unclear; however comparison with histologic studies suggests that these foci may represent subclinical features of lipoprotein extravasation after breakdown of the inner blood–retina barrier. Therefore, it is concluded the foci represent extravasated lipoproteins and/or proteins being a very early subclinical barrier breakdown sign in DME

In contrast, study by Vujosevic S et al & Grisby J.G.et al have stated that HRDs are associated with inflammatory responses in the retina. As the retinal inflammation increases, microglial cells are transformed into an activated state, increasing in number and translocating through the retina. When microglial cells are activated, their morphologies change and they aggregate.(9,10)

At 6 weeks followup post bevacizumab we found 8 patients out of 10(80 %) ( P<0.001) patients with subfoveal HRF had >10 letters BCVA loss. At 3 months follow up 7/12 patients had BCVA loss of > 10 letters( P<0.012).

Thus ,we  found that hyperreflective foci(HRF) located subfoveally  are associated with poor visual prognosis in DME .

As with any retrospective analysis, there are limitations of this study. In particular, a lack of  control group and the small number of eyes studied ,limited angiographic data provides a challenge in accounting for macular ischemia.In our study, we did not investigate the predictive value of other baseline factors, such as phakic status, baseline acuity, retinal thickness.Future prospective studies of these baseline factors are required to confirm the predictive role of the OCT biomarkers we have identified.

Conclusion :

OCT based imaging biomarkers helps us to pick up disease at an early stage, to confirm our diagnosis in case of doubtful, grade the severity of disease and to modify our treatment regimen accordingly.In this study, we examined different  OCT biomarkers and their correlation with functional outcome in DME patients treated with bevacizumab.In conclusion, we identified that Photoreceptor outer segment  thickness , integrity of Ellipsoid Zone , External Limiting Membrane  and location of High Reflective Foci are important OCT biomarkers that predict better visual outcome in DME patients treated with Bevacizumab.

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