BMJ Publishing Group Limited (BMJ) disclaims all liability and ......Taube JM , et al. J Immunother Cancer 2021; 9:e002197. doi: 10.1136/jitc-2020-002197 doi: 10.1136/jitc-2020-002197

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



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



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



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)



E.

CK Cell Densities in Breast TMA



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



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



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



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)



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



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



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)





D.

E.

CD8 Cell Densities in NSCLC TMA



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



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



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



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)



F.

CK Cell Densities in NSCLC TMA



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



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



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



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)



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



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



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)



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.



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,


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



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,


%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,


%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



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,


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



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,


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



Documents

BMJ Publishing Group Limited (BMJ) disclaims all liability and ......Taube JM , et al. J Immunother Cancer 2021; 9:e002197. doi: 10.1136/jitc-2020-002197 doi: 10.1136/jitc-2020-002197