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BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
C.
D.
CD8 Cell Densities in Breast TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.95 (0.84) 0.81 (0.91) 0.76 (1.22) 0.72 (0.81) 0.78 (1.03)
Site 2 N/A 0.83 (0.74) 0.81 (1.45) 0.79(0.96) 0.82 (1.21)
Site 3 N/A 0.95 (1.31) 0.74 (0.78) 0.82 (0.95)
Site 4 N/A 0.76 (0.59) 0.77 (0.69)
Site 5 N/A 0.83 (0.98)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.97 (0.93) 0.88 (0.87) 0.97 (0.95) 0.96 (0.98) 0.94 (1.31) 0.85 (1.11)
FoxP3 Cell Densities in Breast TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.98 (0.84) 0.67 (1.02) 0.80 (0.91) 0.53 (0.89) 0.53 (0.72)
Site 2 N/A 0.74 (1.15) 0.90 (0.92) 0.65 (0.93) 0.61 (0.69)
Site 3 N/A 0.82 (1.14) 0.47 (1.03) 0.51 (0.88)
Site 4 N/A 0.61 (0.94) 0.61 (0.72)
Site 5 N/A 0.57 (0.58)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.89 (0.97) 0.89 (0.94) 0.85 (0.96) 0.89 (0.83) 0.60 (0.74) 0.81 (0.84)
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
E.
CK Cell Densities in Breast TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.94 (0.90) 0.91 (1.06) 0.85 (0.89) 0.80 (1.02) 0.81 (0.77)
Site 2 N/A 0.92 (1.00) 0.82 (0.95) 0.80 (1.11) 0.86 (0.85)
Site 3 N/A 0.88 (1.01) 0.86 (1.18) 0.83 (0.87)
Site 4 N/A 0.79 (1.05) 0.75 (0.79)
Site 5 N/A 0.80 (0.69)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.95 (0.93) 0.97 (0.99) 0.97 (1.04) 0.87 (0.91) 0.89 (0.77) 0.84 (1.15)
F.
PD-1 Cell Densities in Breast TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.95 (0.87) 0.90 (0.75) 0.91 (1.02) 0.82 (0.57) 0.88 (1.68)
Site 2 N/A 0.94 (0.68) 0.94 (0.93) 0.82 (0.51) 0.90 (1.89)
Site 3 N/A 0.97 (0.72) 0.82 (1.13) 0.91 (1.33)
Site 4 N/A 0.81 (1.52) 0.87 (1.76)
Site 5 N/A 0.92 (1.08)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.97 (1.06) 0.99 (1.09) 0.98 (0.98) 0.97 (1.23) 0.77 (0.34) 0.91 (0.52)
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
Supplementary Figure S2. Breast TMA intra- and inter-site cell density concordance analysis.
(A) Representative intra-site cell density concordance plots for each lineage marker, CD68, CD8,
FoxP3, and CK (tumor cells). Detailed inter- and intra-site comparisons are shown for (B) CD68,
(C) CD8, (D) FoxP3, (E) CK, (F) PD-1, and (G) PD-L1. Averages show R2 (slope and standard
deviation of slope). P-values for all concordance values were statistically significant.
G.
PD-L1 Cell Densities in Breast TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.94 (0.98) 0.93 (0.89) 0.88 (0.92) 0.85 (1.05) 0.85 (0.99)
Site 2 N/A 0.93 (0.90) 0.88 (0.93) 0.88 (1.06) 0.90 (1.04)
Site 3 N/A 0.94 (0.92) 0.87 (0.77) 0.89(0.93)
Site 4 N/A 0.79 (0.77) 0.80 (0.91)
Site 5 N/A 0.91 (1.07)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.96 (1.04) 0.99 (1.06) 0.91 (0.98) 0.90 (0.92) 0.47 (0.39) 0.57 (0.48)
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
D.
E.
CD8 Cell Densities in NSCLC TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.72 (0.76) 0.87 (0.71) 0.83 (1.47) 0.78 (1.87) 0.59 (0.39)
Site 2 N/A 0.62 (0.46) 0.71 (1.75) 0.70 (2.61) 0.70 (0.60)
Site 3 N/A 0.91 (1.27) 0.86 (1.36) 0.50 (0.29)
Site 4 N/A 0.74 (1.13) 0.60 (0.26)
Site 5 N/A 0.54 (0.19)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.87 (0.53) 0.83 (0.58) 0.85 (0.61) 0.81 (0.87) 0.84 (0.65) 0.84 (1.12)
FoxP3 Cell Densities in NSCLC TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.86 (1.07) 0.85 (0.98) 0.84 (1.04) 0.72 (0.83) 0.71 (0.80)
Site 2 N/A 0.83 (1.12) 0.91 (0.94) 0.84 (0.77) 0.77 (0.73)
Site 3 N/A 0.83 (1.15) 0.75 (0.96) 0.75 (0.94)
Site 4 N/A 0.72 (0.76) 0.81 (0.81)
Site 5 N/A 0.64 (0.82)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.87 (0.57) 0.88 (0.65) 0.70 (0.48) 0.84 (0.56) 0.88 (0.92) 0.85 (0.95)
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
F.
CK Cell Densities in NSCLC TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.89 (0.94) 0.96 (0.93) 0.95 (0.99) 0.84 (0.90) 0.83 (0.90)
Site 2 N/A 0.96 (0.96) 0.95 (0.97) 0.89 (1.19) 0.87 (0.93)
Site 3 N/A 0.98 (0.95) 0.91 (1.16) 0.88 (0.93)
Site 4 N/A 0.85 (1.15) 0.83 (0.88)
Site 5 N/A 0.88 (0.72)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.90 (0.59) 0.90 (0.63) 0.90 (0.67) 0.81 (0.58) 0.89 (0.59) 0.91 (1.21)
G.
PD-1 Cell Densities in NSCLC TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.96 (0.84) 0.95 (0.96) 0.95 (1.05) 0.86 (0.68) 0.88 (1.25)
Site 2 N/A 0.95 (0.83) 0.96 (0.89) 0.85 (0.57) 0.87 (1.48)
Site 3 N/A 0.98 (0.91) 0.82 (1.24) 0.86 (1.24)
Site 4 N/A 0.82 (1.36) 0.85 (1.35)
Site 5 N/A 0.87 (0.92)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.99 (1.04) 0.99 (1.02) 0.94 (0.69) 0.94 (1.15) 0.93 (1.17) 0.72 (0.2)
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
Supplementary Figure S3. Representative photomicrographs from the NSCLC TMAs and inter-
and intra-site cell density concordances. (A) A NSCLC carcinoma TMA was cut into 12 serial
sections. Two slides were provided to each of the six sites, with one slide stained each of two
days at each site. Images show the serial sections from a representative TMA core stained at each
site over two days and highlight the visual consistency of automated mIF assay staining results.
(B). Average inter- and intra-site concordance plots densities of each cell lineage. Data shown as
R2 (slope and standard deviation (SD) of slope). Detailed inter- and intra-site comparisons are
shown for (C) CD68, (D) CD8, (E) FoxP3, (F) CK, (G) PD-1, and (H) PD-L1. P-values for all
concordance values are statistically significant.
H.
PD-L1 Cell Densities in NSCLC TMA
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Inte
r-s
ite
Site 1 N/A 0.85 (0.87) 0.87 (0.82) 0.93 (0.96) 0.79 (0.86) 0.80 (1.22)
Site 2 N/A 0.83 (0.76) 0.84 (0.86) 0.83 (0.86) 0.82 (1.23)
Site 3 N/A 0.81 (0.79) 0.89 (0.86) 0.90 (1.10)
Site 4 N/A 0.74 (0.88) 0.76 (1.19)
Site 5 N/A 0.90 (1.20)
Site 6 N/A
Concordance, R2 (Slope)
Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Intra-site 0.89 (1.01) 0.87 (0.99) 0.82 (0.89) 0.77 (0.88) 0.77 (0.93) 0.45 (0.32)
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
Batch Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
1
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 19
19 20 21 22 23 24
25 26 27 28 29 30
2
31 36 41 46 51 56
32 37 42 47 52 57
33 38 43 48 53 58
34 39 44 49 54 59
35 40 45 50 55 60
Supplementary Table S1. Tissue section serialization and distribution of unstained
slides of tonsil tissue to the sixlaboratories.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
Supplementary Table S2. Inter-site concordance analysis of %PD-L1 within CK+ and CD68+ cells
in breast TMAs. P-values for all concordance values are statistically significant.
Concordance,
R2 (Slope)Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Site 1 N/A 0.95 (1.01) 0.91 (0.98) 0.79 (0.87) 0.83 (0.85) 0.89 (0.85)
%P
D-L
1 w
ith
in C
K+
cells
Site 2 N/A 0.90 (1.01) 0.72 (0.87) 0.88 (0.90) 0.90 (0.88)
Site 3 N/A 0.81 (0.95) 0.84 (1.01) 0.86 (0.81)
Site 4 N/A 0.67 (0.86) 0.75 (0.83)
Site 5 N/A 0.91 (0.92)
Site 6 N/A
Concordance,
R2 (Slope)Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Site 1 N/A 0.91 (0.94) 0.91 (0.90) 0.85 (0.81) 0.90 (0.93) 0.90 (0.94)
%P
D-L
1 w
ith
in C
D68+
cells
Site 2 N/A 0.86 (0.87) 0.83 (0.77) 0.88 (0.92) 0.89 (0.91)
Site 3 N/A 0.87 (1.04) 0.89 (0.90) 0.86 (0.98)
Site 4 N/A 0.84 (0.79) 0.83 (1.06)
Site 5 N/A 0.92 (0.96)
Site 6 N/A
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
Supplementary Table S3. Inter-site concordance analysis of %PD-L1 within CK+ and CD68+ cells
in NSCLC TMAs. Average inter-site concordance (slope + SD) for %PD-L1 within CK+ cells was
0.89 (0.92 ± 0.06) and for %PD-L1 within CD68 was 0.72 (0.87 ± 0.10). P-values for all
concordance values are statistically significant.
Concordance,
R2 (Slope)Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
%P
D-L
1 w
ith
in C
K+
cells
Site 1 N/A 0.93 (0.97) 0.91 (0.91) 0.94 (0.94) 0.86 (0.83) 0.84 (0.84)
Site 2 N/A 0.88 (0.91) 0.91 (0.92) 0.84 (0.83) 0.83 (0.84)
Site 3 N/A 0.88 (0.93) 0.96 (1.04) 0.93 (0.93)
Site 4 N/A 0.83 (0.98) 0.83 (0.86)
Site 5 N/A 0.95 (1.01)
Site 6 N/A
Concordance,
R2 (Slope)Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
%P
D-L
1 w
ith
in C
D68+
cells Site 1 N/A 0.74 (0.78) 0.80 (1.06) 0.78 (0.84) 0.74 (0.99) 0.71 (0.92)
Site 2 N/A 0.76 (0.94) 0.73 (0.76) 0.71 (0.89) 0.54 (0.73)
Site 3 N/A 0.70 (1.00) 0.79 (0.89) 0.77 (0.84)
Site 4 N/A 0.72 (0.73) 0.60 (0.89)
Site 5 N/A 0.71 (0.82)
Site 6 N/A
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
Supplementary Table S4. Inter-site concordance analysis for PD-1 / PD-L1 proximity in the
breast TMA. P-values for all concordance values are statistically significant.
Concordance,
R2 (Slope)Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Site 1 N/A 0.90 (0.82) 0.82 (0.73) 0.84 (0.97) 0.73 (0.52) 0.77 (0.49)
Site 2 N/A 0.88 (0.66) 0.84 (0.84) 0.76 (0.46) 0.80 (0.44)
Site 3 N/A 0.91 (0.72) 0.78 (1.16) 0.86 (0.65)
Site 4 N/A 0.73 (1.48) 0.77 (0.47)
Site 5 N/A 0.94 (0.85)
Site 6 N/A
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
Supplementary Table S5. Inter-site concordance analysis for PD-1 / PD-L1 proximity in the NSCLC
TMA. Average inter-site concordance (slope + SD) = 0.84 (0.84 ± 0.26). P-values for all concordance
values are statistically significant.
Concordance,
R2 (Slope)Site 1 Site 2 Site 3 Site 4 Site 5 Site 6
Site 1 N/A 0.95 (0.86) 0.87 (0.94) 0.94 (1.07) 0.80 (0.62) 0.80 (0.63)
Site 2 N/A 0.92 (0.86) 0.94 (0.94) 0.82 (0.56) 0.82 (0.57)
Site 3 N/A 0.89 (0.86) 0.77 (1.29) 0.79 (0.62)
Site 4 N/A 0.76 (1.40) 0.79 (0.57)
Site 5 N/A 0.81 (0.89)
Site 6 N/A
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer
doi: 10.1136/jitc-2020-002197:e002197. 9 2021;J Immunother Cancer, et al. Taube JM
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