Supplementary Appendix

Appendix Table A1

Summary of IMPACT assay versions.

Version

2.01

Version 2.02 Version 2.03 Version 7.01

Markers VEGF-A VEGF-A, VEGF-C, PDGF-C,

G-CSF

VEGF-A, VEGF-C,

PDGF-C

VEGF-A

Capture antibody type (for

VEGF-A)

Biotinylated monoclonal antibody 3C5 (IgG) Biotinylated monoclonal antibody 3C5

[F(ab’)2]

Detection antibody Digoxigenin-labelled monoclonal antibody 26503

Sample volume (per test), μL 8 20 20 8

Sample dilution 1:5 1:1 1:1 1:5

Calibration VEGF-A189 VEGF-A189 VEGF-A189 WHO reference standard (VEGF-A165)

Availability Obsolete Obsolete Obsolete Current

F(ab’) = fragment (antigen-binding); G-CSF = granulocyte colony-stimulating factor; Ig = immunoglobulin; IMPACT = immunological multi-

parametric chip technique; PDGF-C = platelet-derived growth factor-C; VEGF-A = vascular endothelial growth factor-A; VEGF-C = vascular

endothelial growth factor-C; WHO = World Health Organization.

1

Appendix Table A2

Baseline characteristics according to baseline pVEGF-A level.

pVEGF-Ahigh pVEGF-Alow

Characteristic Placebo–paclitaxel

(n = 124)

Bevacizumab–paclitaxel

(n = 120)

Placebo–paclitaxel

(n = 118)

Bevacizumab–paclitaxel

(n = 119)

Age, years

Median (range) 57 (28–77) 57 (28–85) 53 (29–76) 54 (32–82)

Age group, years, n (%)

<40 10 (8) 7 (6) 11 (9) 10 (8)

≥40 to 64 90 (73) 83 (69) 85 (72) 82 (69)

≥65 24 (19) 30 (25) 22 (19) 27 (23)

Region, n (%)

Asia 18 (15) 14 (12) 27 (23) 33 (28)

North America/Europe 58 (47) 61 (51) 53 (45) 47 (39)

2

Other 48 (39) 45 (38) 38 (32) 39 (33)

ECOG PS, n (%)

0 65 (52) 58 (48) 76 (65) 65 (55)

1 59 (48) 62 (52) 41 (35) 54 (45)

Missing 0 0 1 0

Median baseline plasma VEGF-A

level, pg/mL (range)

9.61 9.51 2.95 2.86

Hormone receptor status, n (%)

ER and/or PgR positive 106 (85) 100 (83) 97 (82) 100 (84)

ER and PgR negative 18 (15) 20 (17) 21 (18) 19 (16)

Measurable disease at baseline 116 (94) 105 (88) 98 (83) 97 (82)

Previous adjuvant chemotherapy, n

(%)

58 (47) 59 (49) 60 (51) 57 (48)

ECOG PS = Eastern Cooperative Oncology Group performance status; ER = oestrogen receptor; LR/mBC = locally recurrent or metastatic

breast cancer; PR = progesterone receptor; VEGF-A = vascular endothelial growth factor-A.

3

4

Appendix Table A3

Reasons for treatment discontinuation by agent and by treatment group.

Reason for discontinuation, n

(%)

Paclitaxel discontinuation Bevacizumab/placebo discontinuation

Placebo–paclitaxel

(n = 242)

Bevacizumab–paclitaxel

(n = 239)

Placebo–paclitaxel

(n = 242)

Bevacizumab–paclitaxel

(n = 239)

Discontinued 220 (90.9) 206 (86.2) 213 (88.0) 205 (85.8)

Adverse event 48 (19.8) 72 (30.1) 23 (9.5) 53 (22.2)

Death 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4)

Disease progression 121 (50.0) 71 (29.7) 138 (57.0) 87 (36.4)

Patient withdrawal 25 (10.3) 27 (11.3) 25 (10.3) 30 (12.6)

Physician decision 17 (7.0) 21 (8.8) 11 (4.5) 16 (6.7)

Protocol violation 1 (0.4) 1 (0.4) 1 (0.4) 4 (1.7)

Lost to follow-up 0 1 (0.4) 0 1 (0.4)

Non-compliance 1 (0.4) 0 1 (0.4) 0

5

Other 6 (2.5) 12 (5.0) 13 (5.4) 13 (5.4)

6

Appendix Table A4

Systemic anti-cancer therapy administered after disease progression by type of therapy and treatment group (data cut-off 31 July 2015).

No. of patients (%) Placebo–paclitaxel (n = 242) Bevacizumab–paclitaxel (n = 239)

Any 141 (58.3) 115 (48.1)

Bevacizumab 4 (1.7) 3 (1.3)

Endocrine therapya 65 (26.9) 48 (20.1)

Chemotherapyb 110 (45.5) 95 (39.7)

Taxane 21 (8.7) 17 (7.1)

Anthracycline 36 (14.9) 31 (13.0)

Platinum 19 (7.9) 11 (4.6)

Oral fluoropyrimidine 67 (27.7) 57 (23.8)

Otherc 17 (7.0) 8 (3.3)

aIncludes enzalutamide, everolimus, palbociclib, abemaciclib, MLN0128 (TORC1/2 inhibitor), anti-oestrogens, aromatase inhibitors, leuprorelin

acetate, goserelin and sex hormones.

7

bIncludes taxanes, anthracyclines, platinums, oral fluoropyrimidines, fluorouracil, methotrexate, vinorelbine, gemcitabine, cyclophosphamide,

lomustine, mitomycin, eribulin, ixabepilone and irinotecan.

cExcludes bevacizumab, endocrine therapy, chemotherapy and radiotherapy.

8

Appendix Table A5

Adverse events occurring at any grade in ≥10% of patients or at grade ≥3 in ≥2% of patients in either treatment group.

Adverse event, n (%) Placebo–paclitaxel (n = 233) Bevacizumab–paclitaxel (n = 238)

All grades Grade 3 Grade 4 Grade 5 All grades Grade 3 Grade 4 Grade 5

Alopecia 141 (60.5) 0 0 0 136 (57.1) 0 0 0

Nausea 71 (30.5) 0 0 0 96 (40.3) 3 (1.3) 1 (0.4) 0

Epistaxis 47 (20.2) 0 0 0 94 (39.5) 0 1 (0.4) 0

Peripheral sensory neuropathy 84 (36.1) 8 (3.4) 0 0 91 (38.2) 11 (4.6) 0 0

Fatigue 70 (30.0) 4 (1.7) 0 0 86 (36.1) 4 (1.7) 1 (0.4) 0

Diarrhoea 68 (29.2) 1 (0.4) 0 0 86 (36.1) 4 (1.7) 0 0

Neutropenia 48 (20.6) 13 (5.6) 8 (3.4) 0 76 (31.9) 34 (14.3) 13 (5.5) 0

Hypertension 29 (12.4) 9 (3.9) 0 0 71 (29.8) 26 (10.9) 0 0

Constipation 50 (21.5) 1 (0.4) 0 0 66 (27.7) 2 (0.8) 0 0

Decreased appetite 40 (17.2) 3 (1.3) 0 0 53 (22.3) 0 0 0

9

Rash 30 (12.9) 1 (0.4) 0 0 53 (22.3) 0 0 0

Arthralgia 51 (21.9) 0 0 0 51 (21.4) 0 0 0

Peripheral neuropathy 49 (21.0) 2 (0.9) 0 0 47 (19.7) 6 (2.5) 0 0

Headache 40 (17.2) 0 0 0 45 (18.9) 2 (0.8) 1 (0.4) 0

Vomiting 34 (14.6) 4 (1.7) 0 0 44 (18.5) 3 (1.3) 1 (0.4) 0

Stomatitis 25 (10.7) 1 (0.4) 0 0 44 (18.5) 0 0 0

Myalgia 28 (12.0) 0 0 0 41 (17.2) 0 0 0

Peripheral oedema 41 (17.6) 0 0 0 40 (16.8) 1 (0.4) 0 0

Cough 28 (12.0) 0 0 0 39 (16.4) 1 (0.4) 0 0

Anaemia 38 (16.3) 5 (2.1) 0 0 37 (15.5) 11 (4.6) 0 0

Insomnia 19 (8.2) 2 (0.9) 0 0 35 (14.7) 0 0 0

Dyspnoea 31 (13.3) 2 (0.9) 0 2 (0.9) 32 (13.4) 1 (0.4) 0 0

Dysgeusia 21 (9.0) 0 0 0 32 (13.4) 0 0 0

Urinary tract infection 13 (5.6) 1 (0.4) 0 0 32 (13.4) 3 (1.3) 0 0

10

Asthenia 35 (15.0) 1 (0.4) 0 0 31 (13.0) 1 (0.4) 1 (0.4) 0

Pyrexia 27 (11.6) 0 0 0 31 (13.0) 0 0 0

Nail discoloration 16 (6.9) 0 0 0 29 (12.2) 0 0 0

Dizziness 24 (10.3) 1 (0.4) 0 0 25 (10.5) 0 0 0

Back pain 29 (12.4) 2 (0.9) 0 0 24 (10.1) 2 (0.8) 1 (0.4) 0

Dyspepsia 16 (6.9) 0 0 0 24 (10.1) 0 0 0

Proteinuria 26 (11.2) 1 (0.4) 0 0 23 (9.7) 1 (0.4) 0 0

Nasopharyngitis 34 (14.6) 0 0 0 22 (9.2) 0 0 0

White blood cell count decreased 16 (6.9) 5 (2.1) 1 (0.4) 0 20 (8.4) 7 (2.9) 2 (0.8) 0

Aspartate aminotransferase increased 17 (7.3) 2 (0.9) 0 0 20 (8.4) 4 (1.7) 1 (0.4) 0

Leucopenia 14 (6.0) 5 (2.1) 0 0 17 (7.1) 7 (2.9) 0 0

Neutrophil count decreased 20 (8.6) 8 (3.4) 1 (0.4) 0 15 (6.3) 9 (3.8) 1 (0.4) 0

Pulmonary embolism 1 (0.4) 1 (0.4) 0 0 7 (2.9) 6 (2.5) 1 (0.4) 0

11

Febrile neutropenia 1 (0.4) 1 (0.4) 0 0 5 (2.1) 3 (1.3) 2 (0.8) 0

12

Appendix figure legends

Appendix Fig. A1. Treatment effect according to cut-off selected for plasma VEGF-A in the

AVADO trial: (A) Bevacizumab 7.5 mg/kg; (B) Bevacizumab 15 mg/kg.

CI = confidence interval; PFS = progression-free survival.

13

Appendix Fig. A2. Independent review facility-assessed progression-free survival (intent-to-treat population).

HR = hazard ratio.

14