© 2017 American Medical Association. All rights reserved.

Supplementary Online Content

Stahl A, Krohne TU, Eter N. Comparing alternative ranibizumab dosages for safety and

efficacy in retinopathy of prematurity: a randomized clinical trial. Published online January 8,

2018. JAMA Paediatr. doi:10.1001/jamapediatrics.2017.4838

eTable 1. List of inclusion and exclusion criteria for CARE-ROP

eTable 2. List of all infants who did not reach the primary endpoint analysis at 24 weeks

eTable 3. (a) List of reported medical history conditions of all infants who died during the

study. (b) List of all adverse events occurring in infants who died during the study.

eTable 4. List of all protocol deviations (PD) during the course of the study

eTable 5. Primary outcome analysis per patient and per eye (full analysis set).

eTable 6. Hazard ratios of need for rescue treatment.

eTable 7. Efficacy outcomes.

eTable 8. ROP baseline and final outcome values for all patients

eTable 9. Baseline characteristics and staging for both infants receiving rescue treatment

eTable 10. List of all eyes receiving re-treatment

eTable 11. Complete list of all systemic VEGF levels measured in CARE-ROP

eTable 12. Numbers of all (serious) adverse events occurring during the duration of the study.

eTable 13. Oxygen supplementation

eFigure 1. Blood sampling protocol

eFigure 2. APGAR scores of all randomized patients

eFigure 3. Distribution of plus disease for all eyes over the course of the study.

eFigure 4. Physiologic vascularization.

eFigure 5. Systemic VEGF levels.

eFigure 6. Systolic and diastolic blood pressure measurements for all patients.

eFigure 7. Heart rate, oxygen saturation and respiratory rate for all patients

eFigure 8. Development of weight, length and head circumference development

2

eTable 1. List of inclusion and exclusion criteria for CARE-ROP

Inclusion criteria

- bilateral ROP in zone I (stage 1+, 2+, 3+/-, AP-ROP) or ROP in posterior zone II (stage 3+, AP-ROP)*

- legal representatives or their designates willing and able to attend regular study visits with the study infant

- written informed consent to participate in the study

Exclusion criteria - pediatric conditions rendering the infant ineligible to anti-VEGF treatment or to repeated

blood draws as evaluated by a neonatal ICU specialist and a study ophthalmologist

- congenital brain lesions significantly impairing optic nerve function

- severe hydrocephalus with significantly increased intracranial pressure

- ROP stage 4 and 5

- ROP only in peripheral zone II or zone III

- known hypersensitivity to the study drug or to drugs with similar chemical structures - contraindications for an intravitreal injection as listed in ranibizumab summary of product characteristics (SmPC) (1)

- systemic use of anti-VEGF therapeutics - use of other investigational drugs (excluding vitamins and minerals) at the time of enrollment,

or within 30 days or 5 half-lives prior to enrollment, whichever is longer

* Zone I is defined as twice the distance from the optic disc to the fovea measured temporally, posterior zone IIis defined as three times the distance from the optic disc to the fovea measured temporally

ICU = intensive care unit

3

eTable 2. List of all infants who did not reach the primary endpoint analysis at 24 weeks

Premature study discontinuation (deaths)

infant no. 1 infant no. 2 infant no. 3

ranibizumab dose 0.12 mg 0.20 mg 0.20 mg

sex male female male

date of death (days post baseline) 101 161 115

cause of death sepsis cardiac failure renal / liver failure

base

line

char

acte

rist

ics

at b

irth

GA (weeks + days) 23 + 5 27 + 2 25 + 1

weight [g] 550 720 635

length [cm] 31 30 31

head circumference [cm] 21,5 23 22

AP

GA

R

1' 4 6 4

5' 4 7 6

10' 6 8 7

stag

ing

of R

OP

base

line

ROP stage 3 3 3

ROP zone II p II p II p

plus disease* moderate severe moderate

retinal hemorrhage (clock hours both eyes) 5 15 7

vitreous hemorrhage absent absent absent

last

vis

it date of last visit (days post baseline) 101 160 112

ROP stage no ROP no ROP 1

ROP zone fully vascularized avascular retina II a III

plus disease* absent absent mild

All premature study discontinuations were due to death of the infant (one in the 0.12 mg and two in the 0.2 mg group). All deaths occurred at least 101 days (14 weeks) after study treatment and none was suspected to be related to the study treatment. There were no deaths among infants receiving multiple ranibizumab injections.

* classification of plus disease severity as judged by the investigator

4

eTable 3. (a) List of reported medical history conditions of all infants who died during the study. (b) List of all adverse events occurring in infants who died during the study.

a) 0.12 mg ranibizumab

0.20 mg ranibizumab

infant no. 1 infant no. 2 infant no. 3

medical history location active at

study start

medical history location active at study start medical history location active at

study start

Laparotomy due to NEC worsening non-ocular no Anaemia non-ocular yes Anaemia non-ocular Yes

Laparotomy due to NEC worsening non-ocular no Cardiomyopathy non-ocular yes Respiratory

distress syndrome non-ocular Yes

Laparotomy due to NEC worsening non-ocular no Renal failure non-ocular yes Patent ductus

arteriosus non-ocular Yes

Laparotomy due to NEC worsening non-ocular no sleep-wake

disorder non-ocular yes Intestinal obstruction non-ocular Yes

Necrotising enterocolitis

(NEC) non-ocular no Pulmonary

hypertension non-ocular yes Unspecified fever non-ocular No

Neonatal intestinal perforation non-ocular no Hypothyroidism non-ocular yes Staphylococcal

sepsis non-ocular Yes

Neonatal intestinal perforation non-ocular no Osteopenia non-ocular yes Bronchopulmonary

dysplasia non-ocular Yes

Bacterial sepsis (Escherichia coli) non-ocular no Respiratory

failure non-ocular yes Hypothyroidism non-ocular Yes

Neonatal intestinal perforation non-ocular no

Bronchopulmonary dysplasia

non-ocular yes Intestinal dilatation non-ocular Yes

Neonatal intestinal perforation non-ocular no Arterial

hypertension non-ocular yes

Anaemia non-ocular yes

Patent ductus arteriosus non-ocular yes

Intraventricular haemorrhage non-ocular yes

Cholestasis non-ocular yes

5

0.12 mg ranibizumab 0.20 mg ranibizumab

infant no. 1 infant no. 2 infant no. 3

Arterial hypotension non-ocular yes

Bronchopulmonary dysplasia non-ocular yes

Adrenal insufficiency non-ocular yes

Cardiomyopathy non-ocular yes

Thrombocytopenia non-ocular yes

Coagulation disorder non-ocular yes

Infantile apnoea non-ocular no

Bradycardia non-ocular no

6

b) 0.12 mg ranibizumab

0.20 mg ranibizumab

infant no. 1 infant no. 2 infant no. 3

adverse events severity time from

injection to start of AE [d]

adverse events severity time from

injection to start of AE [d]

adverse events severity time from

injection to start of AE [d]

Intestinal fistula moderate 20 Arterial hypertension mild 7 Hyperglycaemia mild 1

Adrenal insufficiency moderate 1 New retinal

haemorrhage mild 70 Hyperinsulinism mild 1

Gastrointestinal dysbacteriosis moderate 48 Constipation mild 32 Arterial

hypotension moderate 74

Pulmonary hypertension severe* 48 Respiratory tract

infection moderate 28 Pulmonary hypertension moderate 62

Bronchial obstruction severe* 48 Worsening of

ROP moderate 91 Pain moderate 79

Arterial hypotension severe 0 Circulatory

collapse severe 41 Increase in

severity of plus disease

moderate 3

Sepsis severe 101 Circulatory collapse severe 128 New retinal

haemorrhages moderate 43

Cardiopulmonary failure severe 160 New occurence

of plus disease moderate 78

Cardiac failure severe 37 Bronchopulm. dysplasia severe 29

Sepsis severe 37 Hepatic failure severe 62

Renal failure severe 112

Ascites severe 101

Abdominal

compartment syndrome

severe 101

Respiratory failure severe 101

* = rated severe but non-serious by the investigator (all other severe events were rated as serious adverse events (SAEs)) Note: None of the occurring adverse events was suspected by the investigator to be related to the study drug.

7

eTable 4. List of all protocol deviations (PD) during the course of the study

Protocol deviations (PDs)

infant deviation category protocol deviation PD severity

0.12

mg

rani

bizu

mab

1 time schedule visit date not within scheduled time frame minor

2 time schedule visit date not within scheduled time frame minor

3 time schedule visit date not within scheduled time frame minor

4

in- / exclusion criteria study assessments before written informed consent minor

time schedule visit date not within scheduled time frame minor

5 study treatment rescue treatment with re-injection and laser treatment minor

6 time schedule visit date not within scheduled time frame minor

0.20

mg

rani

bizu

mab

1 time schedule visit date not within scheduled time frame minor

2 time schedule visit date not within scheduled time frame minor

3 time schedule visit date not within scheduled time frame minor

4 time schedule randomization date not on date of baseline visit minor

5 time schedule visit date not within scheduled time frame minor

6

in- / exclusion criteria study assessments before written informed consent minor

time schedule visit date not within scheduled time frame minor

All PDs were considered minor since they did not impact the primary endpoint. Most PDs were noted in the category “time schedule” and were due to study visits being outside the pre-defined time window. In all these cases the visit schedule was missed by a few days.

8

eTable 5. Primary outcome analysis per patient and per eye (full analysis set).

Full analysis set (FAS)

Incidence of patients without need for rescue treatment

0.12 mg (N=10) n (%) [95%-CI]

0.20 mg (N=9) n (%) [95%-CI]

Total (N=19) n (%) [95%-CI]

Odds Ratio [95%-CI]

All patients 8 (80.0)

[44.4; 97.5] 6 (66.7) [29.9; 92.5]

14 (73.7) [48.8; 90.9]

1.88* [0.26; 13.49]

Gestational age at birth ≤ 25 weeks 4 (66.7) [22.3; 95.7]

4 (80.0) [28.4; 99.5]

8 (72.7) [39.0; 94.0]

0.50 [0.03; 7.99]

Gestational age at birth > 25 weeks 4 (100.0) [39.8; 100.0]

2 (50.0) [6.8; 93.2]

6 (75.0) [34.9; 96.8]

-

Incidence of eyes without need for rescue treatment

0.12 mg (N=20) n (%) [95%-CI]

0.20 mg (N=18) n (%) [95%-CI]

Total (N=38) n (%) [95%-CI]

Odds Ratio [95%-CI]

All patients 17 (85.0)

[54.5; 98.3] 13 (72.2) [38.5; 93.8]

30 (78.9) [54.6; 93.9]

2.18 [0.26; 18.19]

Gestational age at birth ≤ 25 weeks 9 (75.0) [36.8; 96.3]

9 (90.0) [55.5; 99.7]

18 (81.8) [54.4; 96.4]

0.33 [0.02; 5.46]

Gestational age at birth > 25 weeks 8 (100.0) [-; -]

4 (50.0) [10.6; 89.4]

12 (75.0) [35.7; 96.6]

-

p-value for difference between treatment groups per patient (Cochran-Mantel-Haenszel-Test): 0.529 * Odds ratio estimate adjusted for gestational age (Mantel-Haenszel estimate) p-value for difference between treatment groups per eye (Rao-Scott Chi-Square Test): 0.425 Confidence limits and p-values are calculated assuming a stratified cluster sampling design with subjects equivalent to clusters, eyes equivalent to units within clusters, and gestational age equivalent to strata, using the Taylor series linearization method for variance estimation. For percentages, modified Clopper Pearson confidence limits are calculated. N and n refer to the number of eyes, i.e., for N the number of infants times 2. Note: Patients who discontinued the study prematurely (i.e. due to death) are considered as having received rescue treatment in both eyes in the FAS.

Number of patients and eyes without rescue therapy up to week 24 in the full analysis set (FAS). Note that patients who

discontinued the study prematurely due to death are considered as having received rescue treatment in both eyes in the FAS.

The data shows effective treatment in the majority of patients in both groups with no statistical difference between the two

dosages, confirming the data found in the per protocol analysis.

b)b)

9

eTable 6. Hazard ratios of need for rescue treatment.

Per protocol set (PPS)

Hazard ratio of need for rescue treatment Ranibizumab 0.12 mg (N=9) vs. Ranibizumab 0.20 mg (N=7)

Hazard Ratio [95%-CI]

All patients 0.89* [0.06; 14.36]

Gestational age at birth ≤ 25 weeks 0.89 [0.06; 14.36]

Gestational age at birth > 25 weeks -

Result from Cox proportional hazards model of need for rescue treatment, censored at the last study visit or death, with treatment group as predictor * Stratified by gestational age

Full analysis set (FAS)

Hazard ratio of need for rescue treatment Ranibizumab 0.12 mg (N=10) vs. Ranibizumab 0.20 mg (N=9)

Hazard Ratio [95%-CI]

All patients 0.75* [0.05; 11.97]

Gestational age at birth ≤ 25 weeks 0.75 [0.05; 11.97]

Gestational age at birth > 25 weeks -

Result from Cox proportional hazards model of need for rescue treatment, censored at the last study visit or death, with treatment group as predictor * Stratified by gestational age

Hazard ratios stratified by gestational age are comparable between the two dosage groups

a

b

10

eTable 7. Efficacy outcomes

Full analysis set (FAS)

ROP severity, n (%) Last post-baseline assessment ≤ Week 24

Baseline No ROP III , 1 IIa, 1 III , 1+ Total Ranibizumab 0.12 mg (N=20) IIp, 3+ 9 (45.0) 6 (30.0) 0 (0.0) 0 (0.0) 15 (75.0) I, 3+ 3 (15.0) 0 (0.0) 2 (10.0) 0 (0.0) 5 (25.0) Total 12 (60.0) 6 (30.0) 2 (10.0) 0 (0.0) 20 (100.0) Ranibizumab 0.2 mg (N=18) IIp, 3+ 8 (44.4) 5 (27.8) 0 (0.0) 2 (11.1) 15 (83.3) I, 3+ 2 (11.1) 1 (5.6) 0 (0.0) 0 (0.0) 3 (16.7) Total 10 (55.6) 6 (33.3) 0 (0.0) 2 (11.1) 18 (100.0) Total (N=38) IIp, 3+ 17 (44.7) 11 (28.9) 0 (0.0) 2 (5.3) 30 (78.9) I, 3+ 5 (13.2) 1 (2.6) 2 (5.3) 0 (0.0) 8 (21.1) Total 22 (57.9) 12 (31.6) 2 (5.3) 2 (5.3) 38 (100.0) N and n refer to the number of eyes.

Efficacy shift tables for all eyes in the full analysis set (FAS). The majority of eyes (58%) had no ROP at the last available post-baseline visit. Sixteen eyes (42%) had stage 1 ROP in either anterior zone II (5%) or zone III (37%). Plus disease was present at the last available visit in only one patient. This patient died 115 days post baseline and the last visit was at 16 weeks (112 days).

11

eTable 8. ROP baseline and final outcome values for all patients

Note that last visit was at 24 weeks for most patients with the exception of the three infants who died during the study (see comments in table). p = posterior; a = anterior; n.a. = not applicable, OD = right eye, OS = left eye * classification of plus disease severity as judged by the investigator

staging of ROP (OD) staging of ROP (OS)

baseline last visit baseline last visit comment

patient stage zone plus disease* stage zone plus

disease stage zone plus disease* stage zone plus

disease*

0.12

mg

rani

bizu

mab

1 3 I severe no ROP n.a. absent 3 II p moderate no ROP n.a. absent

2 3 II p moderate 1 III absent 3 II p moderate 1 III absent re-treatment both eyes week 10

3 3 II p mild no ROP n.a. absent 3 II p moderate no ROP n.a. absent

4 3 II p mild 1 III absent 3 II p mild 1 III absent

5 3 II p mild no ROP n.a. absent 3 II p mild no ROP n.a. absent

6 3 II p mild no ROP n.a. absent 3 II p mild no ROP n.a. absent

7 3 II p moderate no ROP n.a. absent 3 II p moderate no ROP n.a. absent death week 12

8 3 I moderate 1 II a absent 3 I mild 1 II a absent re-treatment both eyes week 12

9 3 I severe no ROP n.a. absent 3 I severe no ROP n.a. absent rescue right eye laser + rbz wk. 2

10 3 II p mild 1 III absent 3 II p mild 1 III absent

0.20

mg

rani

bizu

mab

1 3 II p mild 1 III absent 3 II p mild 1 III absent

2 3 II p moderate 1 III absent 3 I moderate 1 III absent

3 3 I mild no ROP n.a. absent 3 II p mild no ROP n.a. absent

4 3 II p severe no ROP n.a. absent 3 I severe no ROP n.a. absent re-treatment both eyes wk 6 + 14

5 3 II p moderate 1 III absent 3 II p moderate 1 III absent

6 3 II p moderate no ROP n.a. absent 3 II p moderate no ROP n.a. absent re-treatment both eyes week 8

7 3 II p severe no ROP n.a. absent 3 II p severe no ROP n.a. absent death week 20

8 3 II p moderate no ROP n.a. absent 3 II p moderate no ROP n.a. absent rescue left eye laser week 2

9 3 II p moderate 1 III mild 3 II p moderate 1 III mild death week 16

12

eTable 9. Baseline characteristics and staging for both infants receiving rescue treatment

Rescue Treatments

infant no. 1 infant no. 2

ranibizumab dose 0.12 mg 0.20 mg sex male male

at b

irth

GA (weeks + days) 24 + 1 24 + 5 weight [g] 400 510 length [cm] missing 29 head circumference [cm] missing 22

AP

GA

R 1' 2 2

5' 4 7

10' 5 7

OD OS OD OS

RO

P

base

line ROP stage 3 3 3 3

ROP zone I I II p. II p. plus disease* severe severe moderate moderate retinal hemorrhage (clock hours) absent absent absent absent vitreous hemorrhage absent absent absent absent

RO

P

at r

escu

e date of rescue (days post bsl) 17 n.a. n.a. 14 type of rescue laser + 0.2 mg rbz n.a. n.a. laser ROP stage 4a 1 2 3 ROP zone missing II p. II a. II a. plus disease* absent absent absent moderate

RO

P

at E

OS

ROP stage no ROP no ROP no ROP no ROP Vascularization to ora serrata no yes yes no plus disease* absent absent absent absent

Note that only one eye per infant received rescue treatment. Both rescued eyes had no ROP at the final study visit.

p = posterior; a = anterior * classification of plus disease severity as judged by the investigator

13

eTable 10. List of all eyes receiving re-treatment

Re-treatments

infant no. 1 infant no. 2 infant no. 3 infant no. 4

ranibizumab dose 0.12 mg 0.12 mg 0.20 mg 0.20 mg

sex female male female male

base

line

char

acte

rist

ics

at b

irth

GA (weeks + days) 23 + 3 25 + 0 26 + 3 25 + 1

weight [g] 560 645 1075 550

length [cm] 30 32 35 30.8

head circumference [cm] 18 22.5 24.5 19.5

AP

GA

R

1' 5 2 7 6

5' 5 6 8 7

10' 7 6 8 8

OD OS OD OS OD OS OD OS

stag

ing

of R

OP

base

line

ROP stage 3 3 3 3 3 3 3 3

ROP zone II p II p I I II p I II p II p

plus disease* moderate moderate moderate mild severe severe moderate moderate

retinal hemorrhage (clock hours) 5 6 absent absent 6 6 10 12

vitreous hemorrhage absent absent absent absent absent absent absent absent

re-t

reat

men

t date of re-treatment (days post bsl) 69 105 50 56

ROP stage 3 3 3 3 3 3 3 3

ROP zone II a II a II p II p II p II p II a II a

plus disease* mild mild mild mild moderate moderate mild moderate

re-t

reat

men

t date of 2nd re-treatment (days post 1st re-tx)

71

ROP stage

3 3

ROP zone

II p II p

plus disease*

moderate moderate

end

of s

tudy

ROP stage 1 1 1 1 no ROP no ROP no ROP no ROP

ROP zone III III II a II a n.a. n.a. n.a. n.a.

plus disease* absent absent absent absent absent absent absent absent

vascularization to ora yes yes no no yes yes yes yes

14

Re-treatments were evenly distributed across the two groups. Six eyes received one re-treatment and two eyes received two re-treatments. Final ROP stage at 24 weeks was no ROP in four eyes and stage 1 ROP in four eyes. p = posterior; a = anterior; OD = right eye, OS = left eye * classification of plus disease severity as judged by the investigator

15

eTable 11 a, b. Complete list of all systemic VEGF levels measured in CARE-ROP

Free plasma VEGF (0.12 mg group) patient no.

week 1 2 a 3 4 5 6 7 b 8 c 9 d 10

0 39.32 7.8 * 23.52 23.35 16.18 59.58 30.16 ** 26.68

1 24.18 38.75 41.09 28.83 24.85 30.82 21.52 105.61 30.4 18.51

2 34.9 20.85 28.83

3 33.64 26.18 7.8 * 26.84 31.9 31.15

4 29.49 29.49 30.4 #

5 20.19 7.8 * 25.51 7.8 * 169.39 17.51 18.18 7.8 * 77.66

6 16.84 16.84 19.99 38.55

7

8 20.74

9

10 49.15 #

11 53.89

12 24.18

13 23.52

14

15 23.52 7.8 * #

16 7.8 *

17 22.22

18 17.03

19

20

21

22

* measured value was below detection limit of 15.6 pg/ml and replaced by 7.8 pg/ml

** hemolytic sample (excluded from analysis)

# baseline value before re-injection / rescue

a re-injection in week 10

b patient died

c re-injection in week 15

d unilateral rescue treatment

a)

16

Free plasma VEGF (0.20 mg group)

patient no.

week 1 2 3 4 a 5 6 b 7 c 8 d 9 d, e

0 24,85 46,66 7.8 * 56,52 7.8 * 7.8 * 35,45 7.8 *

1 16,18 63,08 7.8 * 99,82 7.8 * 17,51 22,19 7.8 *

2

16,84

3 22,19 30,82

7.8*

7.8 * 43,85 # **

4

20,19

7.8 * 16,29

5 18,85 63,08

43,37

16,29

6 23,52

20,19

16,84

7

20,19 #

8

7.8 *

7.8 * #

9

16,18

10

11

52,35

12

20,85

26,18

13

27,5

14

22,19

15

16

17

7.8 * #

18

19

20

7.8 *

21

7.8 *

22

23

7.8 *

* measured value was below detection limit of 15.6 pg/ml and replaced by 7.8 pg/ml

** hemolytic sample (excluded from analysis)

# baseline value before re-injection / rescue

a re-injection in week 7 and week 17

b re-injection in week 8

c unilateral rescue treatment

d patient died

e no VEGF measurements available

Note that CTAD buffer was used to prevent thrombolysis. Values represent free plasma VEGF.

b)

17

eTable 12. Numbers of all (serious) adverse events occurring during the duration of the study

Summary of treatment emergent adverse events* Frequency of patients 0.12 mg rbz

(N=10) 0.20 mg

rbz (N=9)

- with adverse event 10 9

- with ocular adverse event 9 9

- with non-ocular adverse event 8 9

- with ocular adverse event with suspected relationship to study treatment 5 a 4 b

- with non-ocular adverse event with suspected relationship to study treatment 0 1 c

- who died 1 2

- who died with suspected relationship to study treatment 0 0

- with serious adverse event 5 6

- with ocular serious adverse event 2 2

- with non-ocular serious adverse event 4 5

- with ocular serious adverse event with suspected relationship to study treatment 1 d 0

- with non-ocular serious adverse event with suspected relationship to study treatment 0 0

* defined as adverse events with start date after the first study treatment a conjunctival haemorrhage, retinal detachment (ROP 4a), injection site haemorrhage b conjunctival haemorrhage, corneal edema, retinal haemorrhage, retinal vascular disorder c respiratory failure, hypotension d retinal detachment (ROP 4a)

Events were distributed evenly between the two study arms.

18

eTable 13. Oxygen supplementation

Supplementary oxygen 0.12 mg (N=10) 0.20 mg (N=9)

N median number of days N median number of

days

befo

re

base

line

intubated 10 23 9 35

CPAP or high flow nasal cannula 10 48 9 39

nasal cannula 2 13 0 -

oxygen by hood / incubator 1 10 0 -

base

line

to

wee

k 24

intubated 6 1.5 8 3.5

CPAP or high flow nasal cannula 8 6 8 12

nasal cannula 5 19 5 83

oxygen by hood / incubator 1 13 0 -

(a) Supplementary oxygen therapy given before and after baseline ranibizumab injection. Before baseline, oxygen demand was evenly distributed between the two groups (more days intubated in the 0.2mg group but more days with CPAP / high flow nasal cannula in the 0.12 mg group). In the post-baseline assessment, the 0.2mg group showed more days with oxygen via nasal cannula compared to the 0.12mg group (83 vs. 19 days). N = number of infants; CPAP = continuous positive airway pressure

(b) Target oxygen ranges (in %) for the first 10 postnatal weeks. Median values and ranges are comparable between the two groups.

Target oxygen saturation (%) - history since birth (median)

postnatal week

0.12 mg (N = 10) 0.20 mg (N = 9)

N lower limit (min-max)

upper limit (min-max) N

lower limit (min-max)

upper limit (min-max)

1 8 85.5 (83.0-86.0) 95.5 (93.0-96.0) 8 85.0 (71.0-88.0) 95.5 (93.0-99.0)

2 8 85.5 (83.0-86.0) 95.5 (93.0-96.0) 8 85.5 (80.0-88.0) 95.5 (93.0-99.0)

3 9 86.0 (80.0-92.0) 96.0 (92.0-98.0) 8 85.5 (80.0-88.0) 95.0 (92.0-98.0)

4 8 85.5 (80.0-86.0) 95.5 (92.0-96.0) 8 85.5 (80.0-88.0) 95.0 (91.0-98.0)

5 8 86.0 (80.0-88.0) 96.0 (92.0-98.0) 7 86.0 (80.0-88.0) 96.0 (93.0-98.0)

6 8 86.0 (80.0-88.0) 96.0 (92.0-98.0) 8 86.0 (80.0-88.0) 96.0 (93.0-98.0)

7 8 86.0 (80.0-88.0) 96.0 (92.0-98.0) 9 88.0 (70.0-88.0) 98.0 (92.0-98.0)

8 10 87.0 (80.0-88.0) 95.5 (92.0-98.0)

8 88.0 (64.0-88.0) 98.0 (93.0-98.0)

9 8 86.0 (80.0-88.0) 96.0 (92.0-98.0) 8 88.0 (80.0-88.0) 98.0 (91.0-98.0)

10 8 86.0 (85.0-88.0) 96.0 (95.0-98.0) 8 88.0 (80.0-90.0) 98.0 (94.0-98.0)

a)

b)

19

eFigure 1. Blood sampling protocol

Flow chart for collection of peripheral blood samples. Whenever possible, blood collection for CARE-ROP was combined with routine clinical blood draws. Care was taken to ensure standardized methods for blood collection and sample handling. CTAD was used as anticoagulant due to its superior qualities for measuring free VEGF. * CTAD = citrate, theophylline, adenosine and dipyridamole.

Blood collection using neonatal cannula.Fill CTAD* collection tubes with 200 - 500 µl of blood.

Close tube and invert 3 times directly after bloodcollection.

Centrifuge within max. 1 hour after blood collection.(3.000 g, 15 min., room temperature).

Use the same centrifuge throughout the study.

Store plasma at -20°C.

20

eFigure 2. APGAR scores of all randomized patients

APGAR scores of all infants are presented as histograms. There was no observable difference between the two treatment groups.

Apgar score - 1 minute

0 1 2 3 4 5 6 7 8 9 100

1

2

APGAR score

nu

mb

er o

f in

fan

ts

Apgar score - 5 minutes

0 1 2 3 4 5 6 7 8 9 100

1

2

3

4

5

APGAR scoren

um

ber

of

infa

nts

Apgar score - 10 minutes

0 1 2 3 4 5 6 7 8 9 100

1

2

3

4

5 ranibizumab 0.12mg

ranibizumab 0.20mg

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eFigure 3. Distribution of plus disease for all eyes over the course of the study.

In almost all eyes plus disease resolved within the first weeks after treatment. There was, however, recurrence of plus disease in some eyes between week 6 and 16. Absent values at later study time points (week 16 – 24) were due to infant deaths.

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eFigure 4. Physiologic vascularization.

Left: Time-to-event analysis for complete vascularization to the ora serrata. Physiologic intraretinal vascularization occurred more effectively in the 0.12 mg group. Note that eyes in which complete vascularization did not occur before the last study visit or before the first rescue or re-injection were censored in this analysis.

Right: Number of patients with complete (top) or at least partial (bottom) vascularization to the ora serrata. In the 0.12 mg group, 50% of patients had complete bilateral vascularization to the ora serrata at week 24. In the 0.2 mg group, complete bilateral vascularization was present in only one patient. Between 67% (0.2 mg group) and 80% (0.12 mg group) of patients showed at least partial bilateral vascularization to the ora serrata (i.e. at least one clock hour of the retina vascularized to the ora serrata).

* patient received bilateral re-treatment

# patient received unilateral rescue treatment

† patient died

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eFigure 5. Systemic VEGF levels.

Free plasma VEGF levels from all patients in the 0.12 mg group (top) and the 0.2 mg group (bottom). Blood samples were collected before (baseline) and during the first six weeks after ranibizumab injection. Horizontal lines represent mean values. CTAD buffer was used to prevent thrombolysis, resulting in generally low VEGF levels since only free plasma VEGF is measured. The dotted gray line represents the detection level of our assay (15.6 pg/ml). Note that several VEGF levels are below detection limit at baseline (i.e. before ranibizumab injection). Only four infants with measurable VEGF levels at baseline show a transient drop of VEGF levels below detection limit during the course of the study (black and blue in the 0.12 mg group; green and orange in the 0.2 mg group). There is no sustained suppression of mean VEGF levels after ranibizumab injection in either group.

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eFigure 6. Systolic and diastolic blood pressure measurements for all patients.

Blood pressure measurements were collected at baseline and during the first six weeks following each ranibizumab injection. Note that the x-axis is not continuous and that values for rescue and re-treatment represent only one to two infants (indicated by brackets). Values at day 0, 1 and 3 for one infant receiving rescue treatment in the 0.2 mg group were not reported.

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eFigure 7. Heart rate, oxygen saturation and respiratory rate for all patients

Measurements were collected at baseline and during the first six weeks following each ranibizumab injection. Note that the x-axis is not continuous and that values for rescue and re-treatment represent only one to two infants (indicated by brackets). Values at day 1 and 3 for one infant receiving rescue treatment in the 0.2 mg group were not reported.

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eFigure 8. Development of weight, length and head circumference development

Charts for weight, length and head circumference development in both study groups are shown over time. Note that the x-axis is not continuous and that values for re-treatments represent only one to two infants (indicated by brackets).

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27

Reference:

1. Food And Drug Administration. Full prescribing information. LUCENTIS (ranibizumab injection) for intravitreal injection [Internet]. 2006 [cited 2017 Sep 3]. Available from: https://www.gene.com/download/pdf/lucentis_prescribing.pdf