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1 Landes Bioscience Supplemental Material to: Martinet et al. High endothelial venules (HEVs) in human melanoma lesions: Major gateways for tumor-infiltrating lymphocytes OncoImmunology 2012; 1(6) article/20492/
2 Supplementary Table S1. Spearman correlation matrix between HEVs and immune parameters HEV number HEV/mm2 CD3 level HEV/mm * CD8 level CD3 level * * CD8 level * * * CD20 level * * * *
3 Supplementary Table S2. Characteristics of melanomas (n=225) according to CD3 + tumorinfiltrating lymphocyte density CD3 Grade 1 Grade 2 Grade 3 HEV /mm2 P < Median (Range) (0.0: 17.5) (0.0: 18.7) (0.0: 27.3) Type SSM 42 (44.2) 23 (43.4) 33 (50.8) p < Autre 17 (17.9) 12 (22.6) 14 (21.5) Acral-lentiginous 24 (25.3) 2 ( 3.8) 1 ( 1.5) Dubreuilh 12 (12.6) 16 (30.2) 17 (26.2) Missing Breslow thickness p < 0.02 <=1mm 43 (47.8) 36 (70.6) 37 (57.8) mm 16 (17.8) 3 ( 5.9) 15 (23.4) >2.0mm 31 (34.4) 12 (23.5) 12 (18.8) Missing Ulceration p = 0.58 No 84 (88.4) 47 (92.2) 55 (85.9) Yes 11 (11.6) 4 ( 7.8) 9 (14.1) Missing Regression p < No 86 (89.6) 38 (74.5) 42 (65.6) Yes 10 (10.4) 13 (25.5) 22 (34.4) Missing Clark Level p < 0.03 I-II 30 (31.6) 22 (43.1) 24 (36.4) III 20 (21.1) 17 (33.3) 23 (34.8) IV-V 45 (47.4) 12 (23.5) 19 (28.8) Missing 4 4 0
4 Supplementary Table S3. Characteristics of melanomas (n=225) according to CD8 + tumorinfiltrating lymphocyte density CD8 Grade 1 Grade 2 Grade 3 HEV /mm2 p < Median (Range) (0.0: 17.5) (0.0: 13.4) (0.0: 27.3) Type p < SSM 40 (43.0) 24 (54.5) 27 (49.1) Others 18 (19.4) 6 (13.6) 14 (25.5) Acral-lentiginous 21 (22.6) 1 ( 2.3) 0 ( 0.0) Dubreuilh 14 (15.1) 13 (29.5) 14 (25.5) Missing Breslow thickness p = 0.17 <=1mm 49 (55.1) 29 (69.0) 31 (57.4) mm 12 (13.5) 7 (16.7) 12 (22.2) >2. mm 28 (31.5) 6 (14.3) 11 (20.4) Missing Ulceration p = 0.20 No 84 (90.3) 40 (95.2) 45 (83.3) Yes 9 ( 9.7) 2 ( 4.8) 9 (16.7) Missing Regression p < No 82 (87.2) 29 (69.0) 35 (64.8) Yes 12 (12.8) 13 (31.0) 19 (35.2) Missing Clark Level p < 0.03 I-II 35 (37.2) 18 (41.9) 21 (37.5) III 19 (20.2) 18 (41.9) 16 (28.6) IV-V 40 (42.6) 7 (16.3) 19 (33.9) Missing 3 3 0
5 Supplementary Table S4. Characteristics of melanomas (n=225) according to CD20 + tumorinfiltrating lymphocyte density CD20 Grade 1 Grade 2 Grade 3 HEV /mm2 p < Median (Range) (0.0: 17.5) (0.0: 18.9) (0.2: 27.3) Type p = 0.09 SSM 70 (47.3) 18 (42.9) 11 (47.8) Others 26 (17.6) 11 (26.2) 5 (21.7) Acral-lentiginous 25 (16.9) 1 ( 2.4) 1 ( 4.3) Dubreuilh 27 (18.2) 12 (28.6) 6 (26.1) Missing Breslow Thickness p = 0.86 <=1mm 84 (58.7) 20 (51.3) 12 (52.2) mm 22 (15.4) 8 (20.5) 4 (17.4) mm 37 (25.9) 11 (28.2) 7 (30.4) Missing Ulceration p = 0.58 Non 132 (89.2) 35 (89.7) 19 (82.6) Oui 16 (10.8) 4 (10.3) 4 (17.4) Missing Regression p < 0.05 Non 123 (82.6) 29 (74.4) 14 (60.9) Oui 26 (17.4) 10 (25.6) 9 (39.1) Missing Clark level p = 0.72 I-II 54 (36.7) 16 (38.1) 6 (26.1) III 42 (28.6) 9 (21.4) 8 (34.8) IV-V 51 (34.7) 17 (40.5) 9 (39.1) Missing 7 1 0
6 Supplementary Table S5 : List of antibodies used in immunofluorescence and immunohistochemistry Antigen Antibody Dilution Species Retrieval method Tissue fixative MECA-79 MECA-79 (BD) 1/2 Rat IgM CD3 SP7 (Abcam) Rabbit IgG CD3 F (Dako) R,F CD8 C8/144B (Dako) 95 C TRS PH high (Dako) F, D CD20 L26 (Beckman coulter) CD31 CD45RO Ab28364 (Abcam) UCHL1 (Dako) Rabbit IgG R,F vwb vwb (Dako) Rabbit IgG R, F DARC Fy6 (BD) R, F ICAM1 1H4 (Abcam) R, F G72 G152 G72 G152 1/10 1/10 Mouse IgM Mouse IgM R R HECA-452 CD45RA HECA-452 (BD) (Beckman coulter) Rat IgM Foxp3 236A/E7 (Abcam) 95 C TRS PH high (Dako) R, F DC-LAMP Fascin Melan-A 1010 E 1.01(Dendritics) 55K2 (Dako) A103 (Dako) Rat IgG 95 C TRS PH high (Dako) 95 C TRS PH high (Dako) F, D R: RCL-2, F: Formalin, D: Dubosc. The HEV-specific antibodies G72 and G152 were kindly provided by Dr Reiji Kannagi (Aichi Cancer Center, Nagoya, Japan).
7 Supplementary Figure S1 Figure S1. Absence of HEV blood vessels in normal skin. Representative pictures of normal skin tissue sections stained with MECA-79 antibody demonstrating the absence of HEV blood vessels in non-pathologic tissues distant from primary melanoma site.
8 Supplementary Figure S2 A HEV number Grade of infiltration : CD3 CD8 CD20 *** *** *** *** *** B C % of samples er of a samples Numb melanoma CD3 CD8 CD20 0 HEV - HEV + HEV - HEV + HEV - HEV + CD3 CD8 CD Grade of infiltration : Grade 1 Grade 2 Grade 3 HEV - HEV + Grade of infiltration : Figure S2. Tumor HEVs predict lymphocyte infiltration in melanoma. (A) Graphs showing the absolute numbers of HEVs in melanoma according to the density of CD3 +,CD8 + and CD20 + TILs. (B) Graphs showing the percentage of melanoma with grade 1 (blue bars), grade 2 (green bars) and grade 3 (red bars) levels of CD3+, CD8+ or CD20+ TILs according to the presence or absence of tumor HEVs. (C) Graphs showing the number of melanoma tumors with grade 1, grade 2 and grade 3 density of CD3+, CD8+ or CD20+ TILs according to the presence (red bars) or absence of tumor HEVs (blue bars). *** p <0.001; Mann Whitney test.
9 Supplementary Figure S3 A /mm2 Spearman r = 0.86 p<0.001 CD3 + cells B CD3 Grade DC-LAMP cluste er Spearman r = 0.92 p< DC-LAMP grade Figure S3 Graphs sho ing the correlation bet een optical grading and a tomatic cell Figure S3. Graphs showing the correlation between optical grading and automatic cell count for CD3 + (A) and DC-LAMP + tumor infiltrating cells (B).
Supplementary Table 1. Antibodies and dilutions used in the immunohistochemical study
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