Background
Methods
Study population
Immunohistochemistry and immunofluorescence
Laser capture microdissection (LCM)-based flow cytometry
Statistical analyses
Results
Patient characteristics and their relationships with TAM cell density, microlocalization, and differentiation patterns
Characteristics | Poor survival group | Extended survival group | p value |
---|---|---|---|
(n = 63)* | (n = 49)* | ||
Age | 0.196 | ||
< 40 | 5 (7.9) | 3 (6.1) | |
40 - 49 | 18 (28.6) | 14 (28.6) | |
50 - 59 | 21 (33.3) | 11 (22.4) | |
60 - 69 | 16 (25.4) | 12 (24.5) | |
≥ 70 | 3 (4.8) | 9 (18.4) | |
Gravidity | 0.576 | ||
0 - 1 | 7 (11.1) | 5 (10.2) | |
2 - 3 | 29 (46.0) | 19 (38.8) | |
4 - 5 | 16 (25.4) | 11 (22.4) | |
≥ 5 | 11 (17.5) | 14 (28.6) | |
Parity | 0.551 | ||
0 - 1 | 42 (66.7) | 30 (61.2) | |
2 - 3 | 21 (33.3) | 19 (38.8) | |
Menopause | 0.789 | ||
Yes | 37 (58.7) | 30 (61.2) | |
No | 26 (41.3) | 19 (38.8) | |
Ascites | 0.382 | ||
Yes | 10 (15.9) | 5 (10.2) | |
No | 53 (84.1) | 44 (89.8) | |
Peritoneal metastasis** | <0.001# | ||
Yes | 25 (39.7) | 5 (10.2) | |
No | 38 (60.3) | 44 (89.8) | |
Lymphatic metastasis | 0.024# | ||
Yes | 19 (30.2) | 6 (12.2) | |
No | 44 (69.8) | 43 (87.8) | |
Stage | <0.001# | ||
I | 6 (9.5) | 21 (42.9) | |
II | 21 (33.3) | 20 (40.8) | |
III | 26 (41.3) | 7 (14.3) | |
IV | 10 (15.9) | 1 (2.0) | |
Histotype | 0.420 | ||
Serous | 38 (60.3) | 26 (53.1) | |
Mucinous | 5 (7.9) | 9 (18.4) | |
Endometrioid | 9 (14.3) | 8 (16.3) | |
Clear cell | 7 (11.1) | 5 (10.2) | |
Undifferentiated | 4 (6.3) | 1 (2.0) | |
Grade | 0.076 | ||
G1 | 9 (14.3) | 15 (30.6) | |
G2 | 28 (44.4) | 21 (42.9) | |
G3 | 26 (41.3) | 13 (26.5) | |
Size of residual site | 0.007# | ||
< 2 cm | 43 (68.3) | 44 (89.8) | |
≥ 2 cm | 20 (31.7) | 5 (10.2) |
Characteristics | Total TAM densities* | M1 TAM densities* | M2 TAM densities* | ||||||
---|---|---|---|---|---|---|---|---|---|
Overall | Islet | Stroma | Overall | Islet | Stroma | Overall | Islet | Stroma | |
Stage |
(p = 0.023)
#
|
(p = 0.206)
|
(p = 0.004)
#
|
(p = 0.162)
|
(p = 0.828)
|
(p = 0.038)
#
|
(p = 0.002)
#
|
(p = 0.008)
#
|
(p = 0.002)
#
|
I | 30.2 ± 18.9 | 16.3 ± 12.1 | 14.0 ± 8.5 | 17.6 ± 12.1 | 10.2 ± 8.0 | 7.4 ± 5.2 | 12.7 ± 7.6 | 6.1 ± 4.5 | 6.6 ± 4.1 |
II | 36.2 ± 23.7 | 17.0 ± 11.1 | 19.2 ± 14.0 | 21.5 ± 14.0 | 11.3 ± 8.3 | 10.2 ± 7.6 | 14.7 ± 10.9 | 5.7 ± 3.4 | 9.0 ± 8.3 |
III | 49.5 ± 32.6 | 22.4 ± 14.7 | 27.0 ± 19.5 | 26.8 ± 19.0 | 12.3 ± 8.8 | 14.4 ± 13.3 | 22.7 ± 16.3 | 10.1 ± 9.1 | 12.6 ± 8.4 |
IV | 52.7 ± 47.7 | 22.4 ± 21.6 | 30.4 ± 24.5 | 23.5 ± 18.8 | 11.2 ± 12.3 | 12.4 ± 9.9 | 29.2 ± 28.4 | 11.2 ± 10.6 | 18.0 ± 18.7 |
Histotype |
(p = 0.049)
#
|
(p = 0.038)
#
|
(p = 0.015)
#
|
(p = 0.016)
#
|
(p = 0.047)
#
|
(p = 0.006)
#
|
(p = 0.193)
|
(p = 0.150)
|
(p = 0.170)
|
Serous | 45.9 ± 33.9 | 22.3 ± 16.0 | 23.6 ± 18.1 | 25.1 ± 16.4 | 13.3 ± 9.8 | 11.9 ± 9.7 | 20.8 ± 18.5 | 9.1 ± 8.4 | 11.7 ± 11.3 |
Mucinous | 45.1 ± 28.2 | 15.1 ± 9.0 | 30.0 ± 20.1 | 27.3 ± 19.8 | 9.8 ± 6.1 | 17.5 ± 14.2 | 17.8 ± 9.8 | 5.3 ± 3.1 | 12.5 ± 7.5 |
Endometrioid | 26.9 ± 15.3 | 13.7 ± 8.4 | 13.2 ± 7.7 | 14.9 ± 10.1 | 8.1 ± 5.9 | 6.8 ± 5.1 | 12.1 ± 6.8 | 5.6 ± 3.4 | 6.4 ± 3.9 |
Clear cell | 26.2 ± 12.0 | 12.6 ± 4.4 | 13.6 ± 7.9 | 12.3 ± 5.0 | 6.8 ± 3.9 | 5.6 ± 2.7 | 13.8 ± 9.6 | 5.8 ± 4.2 | 8.0 ± 6.5 |
Undifferentiated | 34.8 ± 22.6 | 19.8 ± 13.3 | 15.0 ± 10.3 | 21.8 ± 16.0 | 13.2 ± 10.9 | 8.6 ± 5.9 | 13.0 ± 6.9 | 6.6 ± 2.9 | 6.4 ± 4.7 |
Grade |
(p = 0.001)
#
|
(p < 0.001)
#
|
(p = 0.002)
#
|
(p < 0.001)
#
|
(p = 0.002)
#
|
(p = 0.002)
#
|
(p = 0.006)
#
|
(p = 0.003)
#
|
(p = 0.028)
#
|
G1 | 30.2 ± 18.2 | 13.0 ± 8.8 | 17.2 ± 12.3 | 17.6 ± 11.9 | 8.2 ± 6.2 | 9.4 ± 7.7 | 12.6 ± 7.7 | 4.8 ± 3.3 | 7.8 ± 5.9 |
G2 | 34.1 ± 20.6 | 16.7 ± 9.8 | 17.4 ± 12.0 | 18.2 ± 11.5 | 9.9 ± 6.8 | 8.3 ± 6.0 | 15.9 ± 11.1 | 6.8 ± 4.1 | 9.1 ± 7.9 |
G3 | 54.4 ± 37.5 | 25.5 ± 17.8 | 28.9 ± 21.6 | 30.4 ± 19.6 | 15.1 ± 10.8 | 15.3 ± 12.8 | 24.0 ± 20.8 | 10.4 ± 9.9 | 13.6 ± 12.3 |
Size of residual site |
(p = 0.625)
|
(p = 0.650)
|
(p = 0.637)
|
(p = 0.963)
|
(p = 0.497)
|
(p = 0.595)
|
(p = 0.328)
|
(p = 0.077)
|
(p = 0.773)
|
< 2 cm | 39.6 ± 29.6 | 18.6 ± 13.8 | 20.9 ± 17.4 | 22.4 ± 16.0 | 11.6 ± 8.4 | 10.7 ± 10.1 | 17.2 ± 15.6 | 7.0 ± 6.5 | 10.2 ± 10.1 |
≥ 2 cm | 42.8 ± 27.4 | 20.0 ± 14.1 | 22.7 ± 14.9 | 22.2 ± 15.7 | 10.3 ± 8.8 | 11.9 ± 8.6 | 20.6 ± 14.3 | 9.7 ± 8.0 | 10.8 ± 7.8 |
Characteristics
|
Islet/stroma ratio of*
|
Overall ratio of
|
Intra-islet ratio of
|
Intra-stroma ratio of
| |||||
Total TAMs
|
M1 TAMs
|
M2 TAMs
|
M1/M2 TAMs*
|
M1/M2 TAMs*
|
M1/M2 TAMs*
| ||||
Stage |
(p = 0.054)
|
(p = 0.585)
|
(p = 0.406)
|
(p = 0.012)
#
|
(p = 0.106)
|
(p = 0.490)
| |||
I | 1.3 ± 0.7 | 1.7 ± 1.2 | 1.1 ± 0.8 | 1.4± 0.5 | 1.8 ± 0.8 | 1.2 ± 0.7 | |||
II | 1.1 ± 0.6 | 1.5 ± 1.3 | 0.9 ± 0.7 | 1.5± 0.5 | 2.0 ± 0.9 | 1.4 ± 0.7 | |||
III | 0.9 ± 0.3 | 1.2 ± 0.9 | 0.8 ± 0.4 | 1.3± 0.6 | 1.6 ± 0.9 | 1.2 ± 0.7 | |||
IV | 0.9 ± 0.6 | 1.6 ± 2.6 | 1.0 ± 0.9 | 1.0± 0.5 | 1.3 ± 1.0 | 1.1 ± 0.7 | |||
Histotype |
(p = 0.005)
#
|
(p = 0.197)
|
(p = 0.073)
|
(p =0.129)
|
(p = 0.651)
|
(p = 0.377)
| |||
Serous | 1.1 ± 0.6 | 1.6 ± 1.2 | 1.0 ± 0.7 | 1.4± 0.5 | 1.8 ± 0.9 | 1.3 ± 0.7 | |||
Mucinous | 0.6 ± 0.2 | 0.6 ± 0.2 | 0.5 ± 0.3 | 1.5± 0.5 | 1.9 ± 0.6 | 1.5 ± 0.7 | |||
Endometrioid | 1.1 ± 0.6 | 1.6 ± 2.1 | 1.0 ± 0.3 | 1.3± 0.2 | 1.5 ± 0.9 | 1.2 ± 0.7 | |||
Clear cell | 1.0 ± 0.3 | 1.6 ± 1.3 | 1.0 ± 0.5 | 1.1± 0.5 | 1.6 ± 1.2 | 1.0 ± 0.6 | |||
Undifferentiated | 1.5 ± 0.8 | 1.6 ± 0.8 | 1.4 ± 1.0 | 1.6± 0.5 | 1.9 ± 0.9 | 1.5 ± 0.5 | |||
Grade |
(p = 0.671)
|
(p = 0.228)
|
(p = 0.925)
|
(p =0.159)
|
(p = 0.504)
|
(p = 0.231)
| |||
G1 | 1.0 ± 0.7 | 1.2 ± 0.8 | 0.9 ± 0.9 | 1.5± 0.6 | 1.8 ± 0.8 | 1.4 ± 0.7 | |||
G2 | 1.1 ± 0.7 | 1.7 ± 1.7 | 1.0 ± 0.6 | 1.3 ± 0.5 | 1.6 ± 0.8 | 1.1 ± 0.6 | |||
G3 | 1.0 ± 0.5 | 1.3 ± 1.1 | 0.9 ± 0.6 | 1.4± 0.6 | 1.8 ± 1.0 | 1.3 ± 0.7 | |||
Size of residual site |
(p = 0.816)
|
(p = 0.533)
|
(p = 0.025)
#
|
(p =0.059)
|
(p =0.004)
#
|
(p = 0.689)
| |||
< 2 cm | 1.0 ± 0.5 | 1.5 ± 1.2 | 0.9 ± 0.6 | 1.4± 0.5 | 1.9 ± 0.9 | 1.2 ± 0.7 | |||
≥ 2 cm | 1.1 ± 0.7 | 1.3 ± 1.7 | 1.2 ± 0.8 | 1.2± 0.6 | 1.3 ± 0.9 | 1.3 ± 0.7 |
LCM-based flow cytometry validation of the immunofluorescence analysis on TAMs
Kaplan–Meier survival analysis based on the intratumoral distribution patterns of TAMs
Multivariate Cox regression analysis of the independence of TAM-related parameters
Patient groups | Total TAM densities* | M1 TAM densities* | M2 TAM densities* | ||||||
---|---|---|---|---|---|---|---|---|---|
Overall | Islet | Stroma | Overall | Islet | Stroma | Overall | Islet | Stroma | |
Poor survival group | 41.7 ± 30.1 | 19.4 ± 14.3 | 22.3 ± 16.9 | 22.0 ± 15.0 | 10.8 ± 8.1 | 11.2 ± 9.5 | 19.6 ± 17.5 | 8.5 ± 8.4 | 11.1 ± 10.3 |
Extended survival group | 38.6 ± 27.8 | 18.4 ± 13.3 | 20.2 ± 16.8 | 22.7 ± 17.1 | 12.0 ± 9.5 | 10.8 ± 10.1 | 15.9 ± 11.8 | 6.4 ± 4.1 | 9.4 ± 8.7 |
p value# | 0.583 | 0.713 | 0.518 | 0.535 | 0.819 | 0.503 | 0.822 | 0.761 | 0.199 |
Patient groups
|
Islet/stroma ratio of*
|
Overall ratio of
|
Intra-islet ratio of
|
Intra-stroma ratio of
| |||||
Total TAMs
|
M1 TAMs
|
M2 TAMs
|
M1/M2 TAMs*
|
M1/M2 TAMs*
|
M1/M2 TAMs*
| ||||
Poor survival group | 1.0 ± 0.6 | 1.5 ± 1.5 | 0.9 ± 0.7 | 1.3 ± 0.6 | 1.7 ± 1.0 | 1.2 ± 0.7 | |||
Extended survival group | 1.1 ± 0.6 | 1.4 ± 1.1 | 0.9 ± 0.6 | 1.5 ± 0.5 | 1.8 ± 0.7 | 1.4 ± 0.7 | |||
p value | 0.113 | 0.364 | 0.930 | 0.061 | 0.802 | 0.225 |
Parameters | HR | 95% CI | p value |
---|---|---|---|
Age | 0.234 | ||
< 40 | 1 (reference) | - | |
40 - 49 | 1.016 | (0.205, 4.931) | |
50 - 59 | 1.664 | (0.386, 7.181) | |
60 - 69 | 1.239 | (0.292, 5.253) | |
≥ 70 | 0.329 | (0.043, 2.492) | |
Gravidity | 0.726 | ||
0 - 1 | 1 (reference) | - | |
2 - 3 | 1.000 | (0.424, 2.355) | |
4 - 5 | 0.904 | (0.345, 2.367) | |
≥ 5 | 0.612 | (0.184, 2.031) | |
Parity | 0.851 | ||
0 - 1 | 1 (reference) | - | |
2 - 3 | 1.068 | (0.536, 2.129) | |
Menopause | 0.944 | ||
Yes | 1 (reference) | - | |
No | 0.969 | (0.407, 2.304) | |
Ascites | 0.120 | ||
Yes | 1 (reference) | - | |
No | 0.421 | (0.142, 1.252) | |
Peritoneal metastasis | <0.001# | ||
Yes | 1 (reference) | - | |
No | 10.753 | (3.768, 30.685) | |
Lymphatic metastasis | 0.678 | ||
Yes | 1 (reference) | - | |
No | 0.814 | (0.307, 2.156) | |
Stage | <0.001# | ||
I | 1 (reference) | - | |
II | 3.5 | (0.781, 15.677) | |
III | 6.1 | (1.733, 21.469) | |
IV | 38.5 | (7.377, 200.926) | |
Histotype | 0.003# | ||
Serous | 1 (reference) | - | |
Mucinous | 0.119 | (0.017, 0.855) | |
Endometrioid | 0.560 | (0.116, 2.707) | |
Clear cell | 0.780 | (0.146, 4.159) | |
Undifferentiated | 5.952 | (1.339, 26.451) | |
Grade | 0.002# | ||
G1 | 1 (reference) | - | |
G2 | 4.557 | (2.373, 8.753) | |
G3 | 8.197 | (2.456, 27.362) | |
Size of residual site | 0.008# | ||
< 2 cm | 1 (reference) | - | |
≥ 2 cm | 2.786 | (1.307, 5.937) | |
Overall M1/M2 TAM ratio | 0.013# | ||
< 1.371 | 1 (reference) | - | |
≥ 1.371 | 0.448 | (0.238, 0.842) |
Prognostic effects of overall and intra-islet M1/M2 TAM ratios in ovarian cancer histotypes
Ovarian cancer histotypes | Overall M1/M2 TAM ratio | Intra-islet M1/M2 TAM ratio | ||||||
---|---|---|---|---|---|---|---|---|
Mean | HR | 95% CI | p value | Mean | HR | 95% CI | p value | |
Serous | 1.395 | 0.560 | (0.244, 1.286) | 0.172 | 1.802 | 0.576 | (0.333, 0.996) | 0.048# |
Mucinous | 1.550 | 0.756 | (0.465, 0.937) | 0.046# | 1.909 | 0.783 | (0.552, 0.973) | 0.023# |
Endometrioid | 1.264 | 0.923 | (0.913, 0.996) | 0.049# | 1.480 | 0.978 | (0.925, 0.997) | 0.035# |
Clear cell | 1.074 | 0.856 | (0.485, 0.993) | 0.112 | 1.637 | 0.875 | (0.715, 0.982) | 0.042# |
Undifferentiated | 1.632 | 0.712 | (0.587, 0.926) | 0.074 | 1.884 | 0.681 | (0.467, 0.935) | 0.038# |