the authors retrospectively reviewed the medical records of 75 eyes from 75 consecutive patients with exudative amd who were treated only with intravitreal injections of 2.5 mg of bevacizumab between 1 january , 2006 , and 31 june , 2008 , at the vitreoretinal clinic of the inha university hospital , incheon , korea .
the study was performed in accordance with the ethical standards of the declaration of helsinki , and data were gathered after obtaining written informed consent .
this includes consent for the off - label use of bevacizumab as treatment for exudative amd .
approval for this treatment was also obtained from the national ministry of health in south korea .
all patients received follow - up for six months or more , and 54 patients received follow - up for more than 12 months . the potential risks and benefits
were discussed with all patients before they received injections , and all patients read and signed informed consent forms .
inclusion criteria were as follows : 1 ) patients with exudative amd , defined as the presence of active fluorescein leakage from the lesion as seen on fluorescein angiography , who received anti - vegf treatment in at least three initial injections every four weeks ; and 2 ) patients who were followed up for a minimum of six months .
the exclusion criteria included : 1 ) patients diagnosed with polypoidal choroidal vasculopathy ( pcv ) by indocyanine green angiography and treated primarily with pdt ; 2 ) prior macular photocoagulation ; 3 ) a history of subtenon injection of triamcinolone acetonide , or pdt , or anti - vegf within six months before the intravitreal bevacizumab injection ; 4 ) other intraocular surgery or management during follow - up ; 5 ) the presence of coexisting ocular disease causing macular edema ( i.e. , diabetic macular edema , retinal vein occlusion , pseudophakic cystoid macular edema , or uveitis ) ; and 6 ) the presence of comorbid ocular conditions that might affect visual acuity .
thirty - nine patients who underwent pdt more than six months before injection were included in the study .
treatment response was evaluated by comparing the visual acuity ( va ) and central macular thickness ( cmt ) measured by optical coherence tomography ( oct ) at baseline with those same variables in the three follow - up periods : immediately after treatment , six months after treatment , and 12 months after treatment .
the study measured change in va , change in cmt measured by oct , and the number of additional intravitreal bevacizumab injections .
va change was evaluated with consistent methods using the snellen eye examination chart and then converted into a logarithm of the minimal angle of resolution ( logmar ) value .
amd characteristics were evaluated in all patients by clinical examination including indirect dilated fundus examination , color fundus photography , oct , fluorescein angiography , and indocyanine green test , if needed .
greatest linear dimension through fluorescein angiography was calculated by two retinal specialists masked to each genotype . in previous studies   the authors collected peripheral venous blood from all patients for extraction of genomic dna and sequencing of single nucleotide polymorphisms .
genotyping of the snps in the cfh y402h ( rs1061170 ) , loc387715 ( rs10490924 ) and htra1 ( rs11200638 ) genes was carried out using big dye terminator cycle sequencing ( applied biosystems inc . ,
foster city , ca , usa ) on an automated sequencer ( model 3730 , applied biosystems inc . ) .
intravitreal bevacizumab ( avastin ; genentech , south san francisco , ca , usa ) injections of 2.5 mg/0.1 ml were performed after topical anesthesia with 0.5% proparacaine drops ( alcaine ; alcon laboratories , fort worth , tx , usa ) under sterile conditions .
all patients received three initial injections every four weeks and were then treated " as needed " based on if there was sign of recurrence .
in this study , the sign of recurrence was a significant increase ( more than 20% ) of central retinal thickness at foveola due to new or residual subretinal fluid in oct or dye leaks found in fluorescein angiography .
treatment response was evaluated by comparing va and cmt at baseline and after three initial injections during the three follow - up periods ( immediate , six months , and 12 months after treatment ) .
baseline demographic and clinical parameters were compared using the student 's t - test and anova test for continuous variables and chi - square tests for categorical variables .
va improvement and cmt change between baseline and follow - up were evaluated among the different genotypes for each gene at each follow - up period by adjusting for the variables ( age , hypertension and prior pdt ) and using repeated measurement analysis of variance .
the study also used paired t - tests and repeated measurement analysis to evaluate whether undergoing pdt more than six months before the start of study affected va and cmt values at baseline and in each follow - up period .
chicago , il , usa ) . the level of statistical significance was set at p < 0.05 .
the authors retrospectively reviewed the medical records of 75 eyes from 75 consecutive patients with exudative amd who were treated only with intravitreal injections of 2.5 mg of bevacizumab between 1 january , 2006 , and 31 june , 2008 , at the vitreoretinal clinic of the inha university hospital , incheon , korea .
the study was performed in accordance with the ethical standards of the declaration of helsinki , and data were gathered after obtaining written informed consent .
this includes consent for the off - label use of bevacizumab as treatment for exudative amd .
approval for this treatment was also obtained from the national ministry of health in south korea .
all patients received follow - up for six months or more , and 54 patients received follow - up for more than 12 months . the potential risks and benefits
were discussed with all patients before they received injections , and all patients read and signed informed consent forms .
inclusion criteria were as follows : 1 ) patients with exudative amd , defined as the presence of active fluorescein leakage from the lesion as seen on fluorescein angiography , who received anti - vegf treatment in at least three initial injections every four weeks ; and 2 ) patients who were followed up for a minimum of six months .
the exclusion criteria included : 1 ) patients diagnosed with polypoidal choroidal vasculopathy ( pcv ) by indocyanine green angiography and treated primarily with pdt ; 2 ) prior macular photocoagulation ; 3 ) a history of subtenon injection of triamcinolone acetonide , or pdt , or anti - vegf within six months before the intravitreal bevacizumab injection ; 4 ) other intraocular surgery or management during follow - up ; 5 ) the presence of coexisting ocular disease causing macular edema ( i.e. , diabetic macular edema , retinal vein occlusion , pseudophakic cystoid macular edema , or uveitis ) ; and 6 ) the presence of comorbid ocular conditions that might affect visual acuity .
thirty - nine patients who underwent pdt more than six months before injection were included in the study .
treatment response was evaluated by comparing the visual acuity ( va ) and central macular thickness ( cmt ) measured by optical coherence tomography ( oct ) at baseline with those same variables in the three follow - up periods : immediately after treatment , six months after treatment , and 12 months after treatment .
the study measured change in va , change in cmt measured by oct , and the number of additional intravitreal bevacizumab injections .
va change was evaluated with consistent methods using the snellen eye examination chart and then converted into a logarithm of the minimal angle of resolution ( logmar ) value .
amd characteristics were evaluated in all patients by clinical examination including indirect dilated fundus examination , color fundus photography , oct , fluorescein angiography , and indocyanine green test , if needed .
greatest linear dimension through fluorescein angiography was calculated by two retinal specialists masked to each genotype .
in previous studies   the authors collected peripheral venous blood from all patients for extraction of genomic dna and sequencing of single nucleotide polymorphisms .
genotyping of the snps in the cfh y402h ( rs1061170 ) , loc387715 ( rs10490924 ) and htra1 ( rs11200638 ) genes was carried out using big dye terminator cycle sequencing ( applied biosystems inc . ,
foster city , ca , usa ) on an automated sequencer ( model 3730 , applied biosystems inc . ) .
intravitreal bevacizumab ( avastin ; genentech , south san francisco , ca , usa ) injections of 2.5 mg/0.1 ml were performed after topical anesthesia with 0.5% proparacaine drops ( alcaine ; alcon laboratories , fort worth , tx , usa ) under sterile conditions .
all patients received three initial injections every four weeks and were then treated " as needed " based on if there was sign of recurrence . in this study
, the sign of recurrence was a significant increase ( more than 20% ) of central retinal thickness at foveola due to new or residual subretinal fluid in oct or dye leaks found in fluorescein angiography .
treatment response was evaluated by comparing va and cmt at baseline and after three initial injections during the three follow - up periods ( immediate , six months , and 12 months after treatment ) .
baseline demographic and clinical parameters were compared using the student 's t - test and anova test for continuous variables and chi - square tests for categorical variables .
va improvement and cmt change between baseline and follow - up were evaluated among the different genotypes for each gene at each follow - up period by adjusting for the variables ( age , hypertension and prior pdt ) and using repeated measurement analysis of variance .
the study also used paired t - tests and repeated measurement analysis to evaluate whether undergoing pdt more than six months before the start of study affected va and cmt values at baseline and in each follow - up period .
seventy - five patients who were diagnosed with exudative amd were enrolled in the study , and all patients were successfully genotyped using the peripheral blood sample . table 1 shows the demographic and clinical features of exudative amd in the study population .
patient distribution and baseline evaluation , including prior pdt , were described according to each genotype of the cfh y402h , loc387715/htra1 genes . for loc387715 ( rs10490924 ) , eight patients ( 10.7% ) were gg genotype , 27 patients ( 36.0% ) were gt genotype , and 40 patients ( 53.3% ) were tt genotype .
the loc387715 gg genotype had the oldest mean age among the three genotypes ( p = 0.056 ) .
hypertension was also prevalent , with the highest prevalence in the tt genotypes , followed by the gg , and gt genotypes ( p = 0.017 ) .
for the htra1 ( rs11200638 ) polymorphism , similar patient distributions of loc3887715 are seen due to its high linkage disequilibrium with loc387715 .
only one patient had different genotypes in loc387715 ( gg ) and htra1 ( ga ) .
for cfh y402h ( rs1061170 ) , 64 patients ( 85.3% ) were tt genotype , 11 patients ( 14.7% ) were tc genotype , and no patients had the high risk cc genotype .
the cfh y402h tt genotype group had an older mean age than the tc group ( p = 0.139 ) .
the prevalence of hypertension was 53.1% and 18.2% in the tt and tc genotype groups , respectively ( p = 0.036 ) . in both cfh y402h and loc387715 ,
the group with the non - risk homozygous allele had a tendency for higher greatest linear dimension , although it was not statistically significant
. results for patients who had pdt more than six months before bevacizumab treatment did not differ significantly from those who had no history of pdt .
however , the data showed that the high risk group of loc387715/htra1 included more previous pdt patients compared with other groups . in order to compare the bevacizumab treatment response according to genotype in each candidate gene ,
baseline va and cmt were measured and compared with those from the three follow - up periods .
table 2 displays mean va and cmt of patients at baseline and in the three follow - up periods after the initial three injection treatments for each genotype of candidate genes .
mean pretreatment va was 1.175 ( logmar ) and mean pretreatment cmt was 354.5 m for the loc387715 gg genotype ( n = 8) . in the loc387715 gt ( n = 27 ) and tt ( n = 40 ) genotype groups , mean pretreatment va ( p = 0.273 ) and cmt ( p = 0.373 )
mean pretreatment va was 0.946 ( logmar ) and mean pretreatment cmt was 302.3 m for the y402h tt genotype ( n = 64 ) .
for the y402h tc genotype ( n = 11 ) , mean pretreatment va ( p = 0.902 ) was worse but mean pretreatment cmt was improved than in the y402h tt group ( p = 0.868 ) .
this study examined how the variables affect treatment response in the three follow - up periods , by adjusting the variables and using repeated measured analysis for the three follow - up periods compared with the baseline data ( table 3 ) . the treatment response to intravitreal bevacizumab at six months post - treatment improved as the high risk " t " allele increased
; this effect was statistically significant ( logmar ; tt , 0.346 ; gt , 0.264 ; gg , 0.188 ; p = 0.037 ) . however , there was no statistically significant change in va at 12 months after treatment ( p = 0.058 ) among loc3887715 subgroups ( fig .
both tt and tc genotype groups in cfh y402h showed the greatest improvement in va after six months , and the tc group had a more favorable response to treatment although it was not statistically significant ( logmar ; tt , 0.259 ; tc , 0.411 ; p = 0.773 ) . at 12 months after treatment , both the tt and tc genotype groups had decreased va compared to va at six months , but again , this was not statistically significant ( p = 0.859 ) .
bonferroni multiple comparison correction showed that there was no significant relationship between cfh y402h and loc387715 treatment response .
table 4 shows the comparison study of the effect of prior pdt on mean changes in va and cmt , which revealed that prior pdt had no statistically significant effect after bevacizumab injection during the total follow - up period .
however , when compared to the control , va in the pdt group tended to show greater improvements in response to treatment during the follow - up period ( table 5 ) .
this study analyzed the number of additional intravitreal injections of bevacizumab needed to maintain target va or disease stability within subgroups of the cfh y402h and loc387715 genes ( table 6 ) .
the tc genotype group of cfh y402h required additional bevacizumab injections compared to the tt genotype group ( tt , 1.517 ; tc , 3.363 ; p = 0.020 ) . in the subgroup of the loc387715 gene ,
the number of additional injections tended to be higher in the gg genotype group than in other groups ( gg , 2.143 ; gt , 2.000 ; tt , 1.575 ; p = 0.065 ) .
in this retrospective study examining the effects of bevacizumab on exudative age - related macular degeneration in the korean population , patients with the risk t allele of loc387715 had better visual outcome and generally required a lower number of additional injections after the initial three injections .
although the function of loc387715 is unknown , the gene is in nearly complete linkage disequilibrium with the htra1 gene .
the htra1 gene is part of the family of heat shock proteins ( hsp ) [ 45 - 47 ] .
patients with the risk allele of loc387715/htra1 might have a better treatment response because the htra1 polymorphism could cause higher sensitivity to oxidative damage .
the function of hsp is to protect tissues from various types of damage , especially oxidative stress .
the role of hsp is particularly important in the retina , which consumes more oxygen than any other organ in the human body .
hsp protects the retinal pigmented epithelium , the photoreceptor , and even the vascular endothelium .
angiogenesis , which is the main pathogenic factor for exudative age - related macular degeneration , is known to be related to oxidative stress .
thus , if there is an alteration in the protective effect of hsp in the high risk allele group , patients might have sensitivity to oxidative damage and the oxidative stress associated with angiogenesis would then cause much more damage to the retina . accordingly , an anti - vegf agent would be able to inhibit angiogenesis related to the oxidative stress , which might reduce the activity of exudative amd in the retina .
this study hypothesizes that patients with the risk allele in loc387715/htra1 have a better treatment response due to decreased sensitivity to oxidative damage by anti - vegf , which reduces the photoreceptor 's functional loss and increases the chance of recovery against functional alteration of hsp .
therefore , these patients might have better visual acuity and less need for additional injections for disease stability .
visual acuity improved significantly in the risk t allele group during the six - month follow - up period .
similarly , the 12-month results showed a correlation between the t allele and better treatment response , but this correlation was not statistically significant ( p = 0.058 ) . at the end of the entire 12-month follow - up period ,
54 of the original 75 patients remained in the study and 21 patients were lost during follow - up .
patients lost during follow - up after six months were those with tt ( n = 11 ) and gt ( n = 8) genotypes that had good treatment response .
this likely contributed to the less distinguishable differences in treatment response at the end of the study period .
it is still evident that patients with the risk t allele in loc387715 have better visual acuity after the initial treatment with bevacizumab and less need for additional injections .
the anti - angiogenic effect of the treatment may eventually reduce stress , which will have a profound impact on patients with the risk t allele .
patients with the risk c allele in cfh y402h genotype had visual improvement at the final 12-month follow - up .
after the initial three injections of bevacizumab , patients with the risk c allele had better visual acuity .
however , during the monthly follow - up after initial treatment , these patients underwent more additional injections " as needed " based on patient 's symptoms and clinical findings ( tt , 1.517 ; tc , 3.363 injections ; p = 0.020 ) .
finally , patients with the risk c allele had better visual improvement than those with the tt genotype at the 6-month follow - up and at the end of the study period .
interestingly , in the loc387715/htra1 group , patients with a better response received less additional injections in contrast to patients in the cfh group who received more additional injections and also had a better outcome . in order to explain this
, one must consider the pathogenesis of exudative age - related macular degeneration : neovascularization and inflammation .
these two factors are also related to the mechanism underlying the treatment response between different time periods . in the immediate post - treatment phase ,
intravitreal bevacizumab reduces amd activity , such as angiogenesis , and reduces the macular edema .
the immediate post - treatment cmt was reduced to less than 250 m in all groups .
however , in the later phase of treatment ( six months post - treatment ) macular thickness does not seem to be responsible for the change in visual acuity of patients .
all patients had cmt within the normal range at six and 12 months post - treatment , but visual acuity varied between different genotype groups .
thus , it appears that the functional status of the photoreceptor is affected by which pathway ( inflammatory or metabolic ) is responsible for the later phase of the treatment response . with this in mind
, cfh inhibits the alternative pathway , which may cause dysregulation of the common pathway in risk allele group and ultimately may cause continued inflammation . thus ,
additional bevacizumab , an anti - vegf agent with anti - inflammatory effects , is needed to reduce the inflammation . accordingly , appropriate maintenance therapy of additional bevacizumab results in visual improvement .
this may also be applicable to the loc387715/htra1 group , in that immediate post - treatment response did not differ between the various loc387715/htra1 genotypes due to bevacizumab 's initial action in reducing macular edema , regardless of genotype .
but on follow - up at six months , the risk allele group had improved treatment response . on the other hand , in studies of the genetic effect of cfh y402h on caucasian population ,
patients with the c allele required additional injections , but treatment still resulted in worse visual acuity .
this supports the current study 's hypothesis that the altered inflammatory pathway in the cfh polymorphism is responsible for the need for more bevacizumab injections in the risk c allele group .
perhaps if more bevacizumab had been administered after the initial treatment in the caucasian study , there would have been improved visual outcome at the end of study . a recent study by sakurada et al
.   showed that higher t - allele frequency in loc387715 a69s was associated with worse visual prognosis after photodynamic therapy for pcv . on the contrary
, the present study showed that higher t - allele frequency in loc387715 was associated with better visual prognosis after intravitreal bevacizumab for cnv .
these results show the variable effect of genetic polymorphism on treatment response associated with disease phenotype ( pcv vs. cnv ) and treatment modality ( pdt vs. bevacizumab ) .
patients with the risk t - allele could have poor visual prognosis when treated for pcv   but could have a better prognosis when treated for cnv ; also , patients with the risk t - allele could have poor visual prognosis due to pdt but may have an improved visual prognosis on bevacizumab treatment . based on these results , one could logically use genetic information of each disease phenotype to personalize treatment modality . for instance , in pcv patients with the risk t allele , it might be useful to incorporate bevacizumab or bevacizumab / pdt combination therapy rather than pdt treatment alone .
there are several limitations to the present study : the patient cohort is small , this is a retrospective study , and patient follow - up was incomplete , resulting in insufficient 12-month follow - up results . in fact , the results were not statistically significant ; however , genotypically different distributions were observed based on age , diabetes mellitus , and prior pdt in the loc387715/htra1 group . the authors could not rule out that statistical error might be due to small sample size .
therefore , tables 2 and 3 show the adjusted results by multivariate statistical analysis for not only hypertension ( which was statistically significant in the baseline study ) but also for age and prior pdt treatment . in addition , patients who were treated with pdt more than six months before the start of bevacizumab treatment were included .
these patient groups had an unfavorable response to pdt and , therefore , would be potential candidates for bevacizumab treatment .
there was no statistically significant difference in va or cmt between the prior pdt group and the control during follow - up ( table 4 ) .
however , as mentioned previously , statistical error due to limited sample size can not be ruled out .
many of the patients in the high risk group of the loc387715/htra1 group had previously received pdt .
there was also a tendency for enhanced visual improvement over baseline in patients with prior pdt compared to the control .
these results imply that the high risk group , which had a better treatment outcome , might be influenced by pdt .
it appears that this response is a result of pdt / bevacizumab combination therapy , not bevacizumab alone .
finally , this study was conducted on an asian population and may not be directly applicable to caucasian populations . despite these limitations ,
the present study is the first to show statistical relevance between the loc387715/htra1 genes and their pharmacogenetic association to bevacizumab in an asian population .
this study may be a guide to further studies on bevacizumab as well as other anti - vegf agents in asian population .
further studies with prospective randomized , controlled trials are needed , and pharmacogenetic studies of ranibizumab , antioxidant supplements and zinc in asian populations are also recommended .
currently , three monthly initial intravitreal injections of anti - vegf agent and additional maintenance treatment for disease stability is a widely accepted treatment protocol for exudative amd .
however , results are still inconclusive regarding the recommended treatment interval or type of drug used in maintenance treatment .
this report describes a relationship between treatment response and additional injections , although the relationship was not statistically significant .
this study could be a guide for future research examining maintenance treatment intervals according to genotyping in exudative amd patients . in conclusion
, this study showed that different loc387715/htra1 genotypes had different treatment responses when bevacizumab was used to treat exudative amd .
patients with the risk allele had better treatment responses and less need for additional injections .
in addition , patients with the risk allele of cfh y402h needed additional injections of bevacizumab for better visual improvement .
this suggests that shows pharmacogenetic factors could be used to determine treatment modality and dosing and this study may ultimately provide basic data for ' personalized medicine ' in amd .
 purposethe purpose of this study was to determine the pharmacogenetic effects of complement factor h ( cfh ) y402h , loc387715 and high - temperature requirement factor a1 ( htra1 ) genotypes on the treatment of exudative age - related macular degeneration ( amd ) by intravitreal bevacizumab injection in a korean population.methodsseventy-five patients diagnosed with exudative amd were treated with intravitreal bevacizumab ( 2.5 mg ) monotherapy . 
 all patients received three initial intravitreal bevacizumab injections every four weeks and were then treated " as needed " based on clinical findings , optical coherence tomography and fluorescein angiography during the 12 month follow - up period after the third injection.resultsthe difference in visual acuity improvement among the three genotypes of loc387715 were statistically significant at six months post - treatment ( logarithm of the minimum angle of resolution ; tt , 0.346 ; gt , 0.264 ; gg , 0.188 ; p = 0.037 ) . among the loc387715 genotypes , the number of additional injections was lower in patients who had the risk t allele ( gg , 2.143 ; 
 gt , 2.000 ; tt , 1.575 ; p = 0.064 ) . 
 there was no significant difference between visual acuity and central macular thickness change in the cfh y402h polymorphism group during the 12 month follow - up period . 
 however , the tc group of cfh y402h required more additional bevacizumab injections than the tt group ( tt , 1.517 ; tc , 3.363 ; p = 0.020).conclusionsthis study demonstrated that different loc387715/htra1 genotypes resulted in different bevacizumab treatment responses on exudative amd . 
 patients with the risk allele had an improved treatment response and less need for additional injections . 
 however , patients with the cfh y402h risk allele needed more additional injections of bevacizumab in order to improve visual acuity . 
 this study illustrates how pharmacogenetic factors may help determine treatment modality and dosing . 
 this could ultimately provide basic data for ' personalized medicine ' in amd . 
Materials and Methods
Patient and inclusion criteria
Clinical examination
DNA preparation and genotyping
Intravitreal bevacizumab injection
Data analysis
Results
Discussion