Supplementary Table 1. Clinical outcome of original trial population and biomarker population
Clinical outcome / Parental trial population(n=312) / Biomarker population
(n=181)
AT
(n=156) / ATX
(n=156) / P-value† / AT
(n=94) / ATX
(n=87) / P-value†
Response rate*, % / 51% / 69% / 0.01 / 66.7% / 67.8% / 0.25
PFS, months (95% CI) / 8.4 (8.0– 9.0) / 11.2 (10.0– 12.0) / <0.001 / 8.5 (8.1–8.9) / 11.3 (10.0–12.6) / <0.001
OS, months (95% CI) / 23.1 (21.0– 26.8) / 24.2 (21.2– 26.6) / 0.53 / 23.9 (20.8–26.9) / 24.3 (21.6–26.9) / 0.84
*Response rate is based on patientsevaluable for response (parental trial population, n=284; biomarker population, n=169)
†P-value for the comparison between AT and ATX
Supplementary Table 2. Baseline plasma protein levels vs baseline patient and tumor characteristics
VEGF-A, pg/mLMedian (range) / sVEGFR2, pg/mL
Median (range) / ANG2, pg/mL
Median (range) / sTIE2, ng/mL
Median (range) / IL6, pg/mL
Median (range) / IL8, pg/mL
Median (range) / CA9, pg/mL
Median (range) / CA15-3, kU/L
Median (range)
Age
< 55 years
≥ 55 years / 92.8 (2.69 – 1,889)
100 (1.99 – 4,678) / 8,843 (5,708 – 13,430)
8,325 (5,359 – 14,730) / 3,106 (700 – 31,745)
2,769 (1,121 – 10,945) / 24.8 (14.1 – 120)
27.2 (15.4 – 42.4) / 1.11 (0.27 – 23.7)**
1.54 (0.21 – 34.8) / 10.9 (0.82 – 216)
9.8 (2.09 – 188) / 61.7 (14.6 – 2,166)*
85.3 (14.5 –1,473) / 145 (10 – 9,697)
128 (4 – 4,428)
ECOG PS
0
1 / 95.9 (1.99– 1,623)
100 (3.50– 4,678) / 8,778 (5,708 – 13,430)
8,390 (5,359 – 14,730) / 2,704 (700 – 12,316)**
3,152 (1,151 – 31,745) / 24.0 (14.1 – 66.2)**
29.6 (16.0 –120) / 1.18 (0.21 – 12.06)**
1.65 (0.27 – 34.78) / 10.21 (0.82 – 188)
10.80 (2.09 – 216) / 80.4 (14.5 – 1,700)
75.7 (14.6 – 2,166) / 101 (4 – 1,668)*
187 (6 – 9,697)
Hormone receptor
Positive
Negative / 95.5 (2.69– 1,889)
109 (1.99– 4,678) / 8,889 (5,379 – 13,270)
8,520 (5,359 – 14,730) / 2,419 (700 – 7,980)*
3,033 (1,040 – 31,745) / 21.8 (14.1 – 29.1)**
26.1 (15.1 – 120) / 1.19 (0.39 – 5.75)
1.37 (0.21 – 34.78) / 9.78 (2.14 – 65.22)
10.53 (0.82 – 216) / 61.1 (19.2 – 238)
80.4 (14.5 – 2,166) / 159 (4 – 9,697)***
27 (14 – 446)
Measurable disease
Yes
No / 100.1 (1.99– 4,677)
68.3 (3.50– 482) / 8,479 (5,359 – 14,730)
9,011 (6,332 – 12,710) / 2,919 (700 – 31,745)
2,993 (2,162 – 8,867) / 25.6 (14.1 – 120)
22.9 (16.0 – 59.9) / 1.30 (0.27 – 34.78)
1.27 (0.21 – 2.86) / 6.65 (2.09 – 103.71)*
10.84 (0.82 –216) / 82.7 (14.5 – 2,166)
61.1 (20.5 – 153) / 127 (4 – 9,697)
134 (16 – 1,506)
Sites of metastatic disease
Visceral disease
Bone–only disease / 100.5 (1.99 – 4,678)*
57.8 (10.8 – 204) / 8,479 (5,359– 14,730)
9,009 (6,332–11,170) / 2,928 (700– 31,745)
2,925 (1,121– 8,867) / 25.6 (15.1– 120)
23.1 (14.1– 46.8) / 1.34 (0.21– 34.8)
0.93 (0.39– 2.86) / 10.4 (0.82– 215)
9.8 (3.58– 28.7) / 80.8 (14.5– 2,166)
57.8 (29.8– 144) / 129 (4 – 9,697)
156 (20 – 498)
No. of metastatic sites
1
2
≥3 / 87.3 (44.9 – 436)*
76.5 (1.99 – 4,678)
121 (2.69 – 1,889) / 9,009 (6,218 – 13,430)
8,411 (5,359 – 14,730)
8,649 (5,816 – 13,290) / 2,701.55 (1,232 – 8,867)
2,952.2 (1,040 – 12,316)
2,940 (700 – 31,745) / 23.8 (15.6 – 46.8)
25.6 (15.1 – 59.9)
26.0 (14.1 – 120) / 1.45 (0.21 – 3.00)
1.26 (0.27 – 17.67)
1.29 (0.30 – 34.78) / 7.72 (3.07 – 53.59)
9.81 (2.36 – 104)
10.9 (0.82 – 216) / 75.9 (20.5 – 433)
84.1 (14.6 – 1,700)
69.2 (14.5 – 2,166) / 159 (17 – 565)
117 (4 – 4,428)
129 (10 – 9,697)
CA15-3
Low (<25th percentile)
Medium (25–75th percentile)
High (>75th percentile) / 84.1 (1.99 – 4,678)
110 (2.69 – 1,623)
101 (9.39 – 1,889) / 8,477 (5,911 – 13,270)
8,649 (5,816 – 13,430)
8,562 (5,359 – 14,730) / 2,560 (700 – 7,542)
3,069 (1,040– 31,745)
2,942 (1,232– 12,136) / 23.0 (15.4– 48.1)
25.2 (14.1– 59.9)
29.3 (16.2– 120) / 1.19 (0.21 – 5.78)**
1.19 (0.27 – 34.8)
2.1 (0.27 – 23.7) / 8.65 (0.82 – 65.2)**
10.5 (2.09 – 188)
12.9 (4.68 – 216) / 59.4 (14.5– 533)
76.5 (18.0– 1,700)
86.8 (14.6– 1,198) / NA
Significant correlations are shown in bold
*P<0.05;**P<0.01;***P<0.00
Supplementary Table 3. Spearman Rhocorrelation coefficients among protein levels at baseline
VEGF-A (pg/mL) / sVEGFR2 (pg/mL) / ANG2 (pg/mL) / sTIE2 (ng/mL) / IL6(pg/mL) / IL8
(pg/mL) / CA9 (pg/mL)
sVEGFR2 (pg/mL) / 0.01 / - / - / - / - / - / -
ANG2 (pg/mL) / 0.07 / 0.04 / - / - / - / - / -
sTIE2 (pg/mL) / 0.11 / -0.02 / 0.43** / - / - / - / -
IL6 (pg/mL) / 0.27** / -0.05 / 0.37** / 0.23** / - / - / -
IL8 (pg/mL) / 0.39** / -0.13 / 0.28** / 0.16* / 0.29** / - / -
CA9 (pg/mL) / -0.07 / -0.17* / 0.15* / 0.19* / 0.17* / 0.28** / -
CA15-3 (kU/L) / 0.18* / 0.02 / 0.15 / 0.23** / 0.22** / 0.24** / 0.17*
Significant correlations are shown in bold
*P<0.05; **P<0.01
Supplementary Table4. Plasma protein changes vs baseline patient and tumor characteristics
VEGF-A change, %Median (range) / sVEGFR2 change, %
Median (range) / ANG2 change, %
Median (range) / sTIE2 change, %
Median (range) / IL6 change, %
Median (range) / IL8 change, %
Median (range) / CA9 change, %
Median (range)
Age
< 55 years
≥ 55 years / -93.3 (-100 – 618)
-91.5 (-100 – 173) / 4.62 (-24.4 – 43.2)
6.39 (-30.2 – 46.6) / -20.5 (-72.2 – 105)
-18.0 (-63.1 – 78.5) / -8.90 (-46.2 – 54.2)
-12.5 (-39.2 – 31.6) / -16.9 (-94.0 – 2,416)
-11.1 (-92.7 – 1,450) / -14.4 (-91.9 – 914)
-10.6 (-78.0 – 411) / 31.4 (-82.9 – 311)
30.6 (-75.6 – 608)
ECOG PS
0
1 / -92.8 (-100 – 618)
-91.5 (-100 – 41.4) / 4.18 (-21.5 – 46.6)
6.23 (-30.2 – 36.2) / -18.09 (-67.4 – 105)
-18.2 (-72.2 – 76.1) / -8.90 (-46.2 – 54.2)
-13.2 (-44.0 – 33.1) / -7.29 (-82.2 – 1,051)
-20.7 (-94.0 – 2,416) / -12.5 (-91.9 – 415)
-10.8 (-81.1 – 913) / 30.8 (-82.9 – 608)
31.0 (-75.6 – 478)
Hormone receptor
Positive
Negative / -92.3 (-100 – 150)
-95.7 (-100 – 618) / 4.98 (-30.2 – 46.6)
7.62 (-18.3 – 35.4) / -18.4 (-72.2 – 105)
2.15 (-53.7 – 68.6) / -12.8 (-46.2 –54.2)**
2.32 (-24.7 –25.1) / -19.1 (-94.0 – 2,416)
13.2 (-77.8 – 1,051) / -10.5 (-81.1 – 151)
-11.2 (-91.9 – 914) / 32.2 (-82.9 – 608)
19.3 (-74.3 – 311)
Measurable disease
Yes
No / -93.0 (-100 – 618)
-90.0 (-98.0 – 41.4) / 4.92 (-24.4 – 46.6)
8.84 (-30.2 – 25.3) / -18.1 (-72.2 – 105)
-18.2 (-62.2 – 16.9) / -10.4 (-46.2 – 54.2)
-14.2 (-34.0 – 31.6) / -11.2 (-94.0 – 2,416)
-29.5 (-68.6 – 147) / -14.8 (-91.9 –415)*
14.4 (-27.6 – 913) / 29.7 (-82.9 – 608)
38.6 (-70.6 – 289)
Sites of metastatic disease
Visceral disease
Bone–only disease / -92.9 (-100 – 618)
-89.3 (-98.0 – -65.2) / 4.98 (-30.2 – 46.6)
9.97 (-12.5 – 24.2) / -18.0 (-72.2 – 105)
-28.79 (-46.9 –4.46) / -10.5 (-46.2 – 54.2)
-12.0 (-34.0 –4.66) / -12.5 (-94.0 – 2,416)
-34.0 (-68.6 – 68.6) / -10.8 (-91.9 – 415)
-11.3 (-27.6 – 914) / 30.2 (-82.7 – 608)
44.3 (-70.6 – 113)
No. of metastatic sites
1
2
≥3 / -92.0 (-98.0 –-65.2)*
-87.4 (-100 – 618)
-94.6 (-100 – 51.0) / 7.71 (-12.5 – 37.3)
3.68 (-30.2 – 36.0)
5.14 (-24.4 – 46.6) / -17.2 (-63.1 – 23.1)
-19.9 (-67.4 – 61.5)
-17.5 (-72.2 – 105) / -6.95 (-34.0 –23.6)*
-13.2 (-46.2 –31.8)
-7.88 (-44.3 –54.2) / -32.1 (-68.6 – 188)
4.46 (-92.7 – 1,010)
-14.3 (-94.0 – 2,416) / -7.00 (-64.1 – 914)
-17.7 (-71.7 – 415)
-10.7 (-91.9 – 411) / 23.7 (-74.3 – 335)
49.1 (-82.7 – 423)
20.3 (-75.6 – 608)
CA15-3
Low (<25th percentile)
Medium (25–75th percentile)
High (>75th percentile) / -93.5 (-100 – 173)
-93.0 (-100 – 150)
-91.4 (-100 –-5.39) / 6.16 (-20.1 – 35.4)
6.39 (-24.4 – 46.6)
3.23 (-30.2 – 34.3) / -6.75 (-63.1 –105)*
-20.7 (-72.2 – 76.1)
-17.4 (-66.4 –78.5) / -5.60 (-34.5 – 31.8)
-10.8 (-46.2 – 54.2)
-13.1 (-42.8 – 25.1) / -1.45 (-92.7 –1,051)*
-4.98 (-77.8 –2,416)
-44.9 (-94.0 –572) / 3.20 (-76.2 – 914)
-16.8 (-91.9 – 415)
-14.5 (-70.1 – 112) / 37.0 (-74.3 – 608)
29.7 (-82.9 – 478)
25.0 (-57.4 – 597)
Significant correlations are shown in bold
*P<0.05;**P<0.01
Supplementary Table 5.Change in levels of proteins per treatment arm
Baseline
Median (Range) / C2D1
Median (Range) / % change from baseline / P-value* / Baseline
Median (Range) / C2D1
Median (Range) / % change from baseline / P-value*
VEGF-A (pg/mL) / 111 (3.50 – 4,678) / 8.24 (0 – 25.4) / -93.3 / <0.001 / 91.4 (1.99 – 1,889) / 8.27 (0 – 75.5) / -91.4 / <0.001
sVEGFR2 (pg/mL) / 8,426 (5,379 – 14,7) / 9,132 (4,670 – 14,90) / 4.62 / 0.001 / 8,950 (5,359 – 13,430) / 9,047 (5,770 – 14,780) / 5.94 / 0.001
ANG2 (pg/mL) / 2,817 (1,130 – 10,945) / 2,236 (833 – 9,461) / -20.3 / <0.001 / 2,925 (700 – 31,745) / 2,517 (863 – 8,833) / -13.8 / <0.001
sTIE2 (ng/mL) / 25.1 (14.1 – 46.8) / 21.8 (12.3 – 46.5) / -11.8 / <0.001 / 25.5 (15.4 – 120) / 23.3 (15.0 – 68.7) / -8.72 / <0.001
IL6 (pg/mL) / 1.29 (0.27 – 34.8) / 1.39 (0.12 – 122) / 1.66 / 0.526 / 1.29 (0.21 – 12.2) / 1.15 (0.12 – 7.67) / -19.2 / 0.034
IL8 (pg/mL) / 10.2 (0.82 – 87.4) / 9.46 (2.03 – 97.5) / -5.55 / 0.339 / 9.97 (2.14 – 216) / 9.0 (2.13 – 89.2) / -19.4 / 0.020
CA9 (pg/mL) / 72.0 (18.9 – 1,700) / 93.5 (19.3 – 1,001) / 20.4 / 0.047 / 75.9 (14.5 – 1,198) / 104 (16.6 – 610) / 36.2 / <0.001
*Wilcoxon Signed-Rank test
Supplementary Table 6.Spearman Rhocorrelation coefficients among protein changes on C2D1 from baseline
VEGF-A change (%) / sVEGFR2 change (%) / ANG2 change (%) / sTIE2 change (%) / IL6 change (%) / IL8 change (%)sVEGFR2 change (%) / -0.14 / - / - / - / - / -
ANG2 change (%) / -0.04 / 0.08 / - / - / - / -
sTIE2 change (%) / 0.00 / 0.08 / 0.45** / - / - / -
IL6 change (%) / 0.07 / -0.20* / 0.16 / 0.16 / - / -
IL8 change (%) / -0.01 / 0.15 / 0.23** / 0.07 / 0.22** / -
CA9 change (%) / -0.03 / 0.00 / 0.16 / 0.08 / 0.04 / 0.17*
Significant correlations are shown in bold
*P<0.05; **P<0.01
Supplementary Figure 1A. A plot of Martingale residual vs log-transformed CA9 change for death
Black line represents the smoothed lowess curve.
Supplementary Figure 1B. Estimation of an optimal cut-off point for CA9 change
Supplementary Figure 1C. Changes in CA9 levels in the subgroups of a high rise and a low/no rise